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Transcriber’s notes:

In this transcription, paired _underscores_ denote _italicised text_. A
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character is subscripted (mostly in chemical formulae, e.g. CO_{2}).
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  an Medndel —> and Mendel
  akaline —> alkaline
  Wochenscrift —> Wochenschrift
  by —> bei
  transferrence —> transference
  quantitity —> quantity
  recticulin —> reticulin
  sythetical —> synthetical
  Elaatin —> Elastin
  hiaton —> histon




[Illustration: Cover]




                          CARTWRIGHT LECTURES

                                 1894




                                  ON

                         DIGESTIVE PROTEOLYSIS


                     BEING THE CARTWRIGHT LECTURES

                               FOR 1894


        DELIVERED BEFORE THE ALUMNI ASSOCIATION OF THE COLLEGE
                OF PHYSICIANS AND SURGEONS OF NEW YORK.


                                  BY

                        R. H. CHITTENDEN, Ph.D.

       _Professor of Physiological Chemistry in Yale University_


                           NEW HAVEN, CONN.:
                TUTTLE, MOREHOUSE & TAYLOR, PUBLISHERS.
                                 1895




        THE TUTTLE, MOREHOUSE & TAYLOR PRESS, NEW HAVEN, CONN.




PREFACE


The present volume, as explained by the title, consists mainly of
a reprint of the Cartwright Lectures for 1894. These lectures were
originally printed in the current numbers of the Medical Record, but so
many requests have been made for their publication in a more convenient
and accessible form that they are now re-issued, through the courtesy
of the publishers of the Record, in book-form.

It is hoped that these lectures may prove of value not only in
calling attention to some of the fundamental chemico-physiological
facts of digestion, but in stimulating closer investigation of the
many questions which are so intimately associated with a proper
understanding of the processes concerned in the digestion and
utilization of the proteid food-stuffs.

    R. H. CHITTENDEN.




CONTENTS.


  LECTURE I.

  _The general nature of Proteolytic Enzymes and of Proteids._

  Introductory observations,                                           1

    Early history of gastric digestion,                                3

    The proteolytic power of the pancreatic juice,                     7

  The general nature of proteolytic enzymes,                           8

    Origin of proteolytic enzymes,                                     8

    Preparation of pepsin,                                            10

    Reactions and composition of proteolytic enzymes,                 13

    The proteid nature of enzymes,                                    15

    Conditions modifying the action of enzymes,                       17

    The influence of temperature on proteolytic action,               18

    The influence of acids, alkalies, and other substances on the
      activity of enzymes,                                            20

    Action of chloroform on pepsin,                                   21

    Theories of enzyme action with special reference to catalysis,    22

  The general nature of Proteids,                                     27

    Classification of proteids,                                       29

    Chemical composition of some of the more prominent proteids
      occurring in nature,                                            31

    Chemical constitution of proteids,                                33

    The presence of hemi- and anti-groups in all typical proteids,    34

    Cleavage of the albumin-molecule with dilute sulphuric acid,      34

    Hydration and cleavage of albumin by the action of superheated
      water, with formation of atmid-albumoses, etc.,                 37

    Action of powerful hydrolytic agents on proteid matter,           39

    Initial action of pepsin-acid on proteids,                        40

    Scheme of the general line of proteolysis as it occurs in
      pepsin-digestion, with a view to the structure of the
      albumin-molecule,                                               41


  LECTURE II.

  _Proteolysis by pepsin-hydrochloric acid, with a consideration
  of the general nature of proteoses and peptones._

  Proteolysis by pepsin-acid,                                         44

    Formation of hydrochloric acid in the gastric glands,             45

    Liebermann’s theory regarding the formation of the acid of the
      gastric juice,                                                  46

    Differences in the action of free and combined acid,              47

    Proteolysis in the presence of combined acid,                     49

    The combining power of various forms of proteid matter with
      hydrochloric acid,                                              51

    Quantitative estimation of the affinity of the products of
      digestion for acid,                                             53

    Richet’s theory regarding the conjugate character of the acid
      of the gastric juice,                                           54

    Proteolysis in the presence of amido-acids,                       55

    Necessity for knowing the amount of combined acid in the
      stomach-contents,                                               57

    Antiseptic action of the hydrochloric acid of the gastric juice,  58

    The maximum action of pepsin exerted only in the presence of
      free hydrochloric acid,                                         59

    Division of the products of pepsin-proteolysis into three main
      groups,                                                         60

    Detection of the products of digestion,                           61

    Separation of proteoses and peptones from a digestive mixture or
      from the stomach-contents,                                      62

    Some of the chemical properties of peptones,                      64

    The so-called propeptone a mixture of proteoses,                  65

    Pepsin-proteolysis synonymous with a series of progressive
      hydrolytic changes,                                             66

    Chemical composition of proteoses and peptones,                   67

    Pepsin-proteolysis a true hydrolytic and cleavage process,        71

    Schützenberger’s results on the formation of fibrin-peptone,      72

    Amphopeptones the final products of gastric digestion, but
      proteolysis never results in complete peptonization,            73

    Solution of a proteid by pepsin-acid not synonymous with
      peptonization,                                                  75

    Influence of the removal of the products of digestion on the
      activity of the ferment,                                        75

    Lack of complete peptonization by pepsin-acid not due to
      accumulation of the products of digestion,                      76

    The diffusibility of proteoses and peptones,                      77

    Absorption of peptones from the living stomach,                   79

    Differences between natural digestion in the stomach and
      artificial proteolysis,                                         80

    Relative formation of proteoses and peptones in the living
      stomach,                                                        81

    Gastric digestion merely a preliminary step in proteolysis,       81

    Intestinal digestion alone capable of accomplishing all that is
      necessary for the complete nourishment of an animal,            82

  Some physiological properties of proteoses and peptones,            83

    The experiments of Schmidt-Mülheim and Fano on the action of
      peptones when injected into the blood,                          84

    Physiological action of albumoses,                                85

    Introduction of albumoses into the blood,                         87

    Proteose-like nature of the poisons produced by bacteria,         89

    The acrooalbumoses formed by the tubercle-bacillus,               90

    Toxic nature of proteoses and peptones,                           91


  LECTURE III.

  _Proteolysis by trypsin--Absorption of the main products
  of proteolysis._

  Proteolysis by trypsin,                                             93

    Comparison of pepsin and trypsin,                                 94

    Trypsin especially a peptone-forming ferment,                     95

    The primary products of trypsin-proteolysis,                      95

    Scheme of trypsin-digestion, showing the relationship of
      the products formed,                                            96

    The fate of hemi-groups in trypsin-proteolysis,                   97

    The primary products of trypsin-digestion mainly antibodies,      98

    Character and composition of antipeptones,                        99

    Antialbumid as a product of pancreatic digestion,                100

    The peculiar action of trypsin in the formation of amido-acids,
      etc.,                                                          101

    Formation of lysin and lysatin in pancreatic digestion,          103

    The relationship of lysatin to urea,                             105

    Formation of tryptophan or proteinochromogen by trypsin,         105

    Appearance of ammonia in trypsin-proteolysis,                    107

    Relationship between artificial pancreatic digestion and
      proteolysis in the living intestine,                           109

    Leucin and tyrosin products of the natural pancreatic digestion
      in the intestine,                                              112

    The physiological significance of leucin and tyrosin,            113

  Absorption of the main products of proteolysis,                    116

    Absorption of acid-albumin, alkali-albuminate, etc.              117

    Absorption limited mainly to the intestine, very little
      absorption from the stomach,                                   119

    The change which the primary products of proteolysis undergo
      in the process of absorption,                                  120

    Peptones not present in the circulating blood,                   121

    The change which peptones and proteoses undergo by contact with
      the living mucous membrane of the small intestine,             122

    Retrogression of peptones by contact with other living cells,
      etc.,                                                          125

    Functional activity of leucocytes in absorption,                 128

    Digestive leucocytosis incited by nuclein,                       131

    Shore’s experiments on the ability of lymph-cells to assimilate
    either proteoses or peptones,                                    133

    Lymph a true secretion from the blood-vessels,                   134

    Direct excitatory effect of peptones when present in the blood
      on the endothelial cells,                                      136

    Selective activity of endothelial cells,                         137




DIGESTIVE PROTEOLYSIS




LECTURE I.

THE GENERAL NATURE OF PROTEOLYTIC ENZYMES AND OF PROTEIDS.


INTRODUCTORY.

In digestive proteolysis we have a branch of physiological study which
of late years has made much progress. Chemistry has come to the aid
of physiology and by the combined efforts of the two our knowledge of
the digestive processes of the alimentary tract has been gradually
broadened and deepened. That which at one time appeared simple
has become complex, but increasing knowledge has brought not only
recognition of existing complexity, but has enabled us, in part at
least, to unravel it.

By digestive proteolysis is to be understood the transformation of the
proteid food-stuffs into more or less soluble and diffusible products
through the agency of the digestive juices, or more especially through
the activity of the so-called proteolytic ferments or enzymes contained
therein; changes which plainly have for their object a readier and more
complete utilization of the proteid foods by the system.

In selecting this topic as the subject for this series of Cartwright
Lectures I have been influenced especially by the opinion that both for
the physiologist and the physician there are few processes going on in
the animal body of greater importance than those classed under the head
of digestion. Further, few processes are less understood than those
concerned in this broad question of digestive proteolysis, especially
those which relate specifically to the digestion of the various classes
of proteid food-stuffs, and to the absorption and utilization of the
several products formed. Moreover, the subject has ever had for me a
strong attraction as presenting a field of investigation where chemical
work can advantageously aid in the advance of sound physiological
knowledge; and certainly every line of advance in our understanding of
the normal processes of the body paves the way for a better and clearer
comprehension of the pathological or abnormal processes to which the
human body is subject.

You will pardon me if I specially emphasize in this connection
the fact that advance along the present lines was not rapid until
physiologists began to appreciate the importance of investigating
the chemico-physiological problems of digestion by accurate chemical
methods. Something more than simple test-tube study, or even
experimental work on animals, is required in dealing with the changes
which complex proteids undergo in gastric and pancreatic digestion. The
nature and chemical composition of the proteids undergoing digestion,
as well as of the resultant products, are necessary preliminaries to
any rightful interpretation of the changes accompanying digestive
proteolysis; but physiology has been slow to appreciate the
significance of this fact, and, until recently, has done very little
to remedy the noticeable lack of accurate knowledge regarding the
composition and nature of the proteid and albuminoid substances which
play such an important part in the life-history of the human organism,
either as food or as vital constituents of the physiologically active
and inactive tissues. This is to be greatly deprecated, since our
understanding of the nature of proteolysis, of the mode of action of
the enzymes or ferments involved, and of the relationships of the
products formed, is dependent mainly upon an accurate determination of
the exact changes in chemical composition which accompany each step in
the proteolytic process. How otherwise can we hope to attain a proper
appreciation of the real points of difference between bodies so closely
related as those composing the large group of proteids and albuminoids?
Surely, in no other way can we measure the nature or extent of the
changes involved in the various phases of proteolysis than by a
thorough study of chemical composition and constitution, as well as of
chemical reactions and general properties.

In the early history of physiology there was, quite naturally,
little or no thought given to the nature of proteolytic changes. The
gastric juice, as one of the first digestive fluids to be studied,
was recognized as a kind of universal solvent for all varieties of
food-stuffs, and this even long before anything was known regarding
its composition, but beyond this point knowledge did not extend.
Active study of the gastric juice, as you well know, dates from 1783,
when the brilliant Italian investigator Spallanzani commenced his
work on digestion. The names of Carminati, Werner and Montégre[1] are
also associated with various phases of work and speculation in this
early history of the subject, especially those which pertained to the
possible presence of acid in the stomach juices. In 1824, however,
Prout showed conclusively that gastric juice was truly acid, and,
moreover, that the acidity was due to the presence of free hydrochloric
acid, and not to an organic acid. Still, many observations failed to
show the presence of an acid fluid in the stomach, and it was not until
Tiedemann and Gmelin’s[2] masterly researches were published that the
cause of this discrepancy was made clear. It was then seen that the
secretion of an acid gastric juice was dependent upon stimulation or
irritation of the mucous membrane of the stomach, and that so long as
the stomach was free from food or other matter capable of stimulating
the mucosa, it contained very little fluid, and that neutral or very
slightly acid in reaction. These early observers also recorded the fact
that the amount or strength of acid increased with the outpouring of
the secretion, incidental to natural or artificial stimulation, thus
giving a hint of the now well-known fact that any and every secretion
may show variations in composition incidental to the character and
extent of the stimulation which calls it forth.

[1] See Berzelius’s Lehrbuch der Chemie, Band 9, p. 205, 4te Auflage,
for an account of these early discoveries.

[2] Tiedemann und Gmelin: Die Verdauung nach Versuchen. Heidelberg und
Leipzig. 1826.

The period between 1825 and 1833 was characterized especially by the
presentation of the many results bearing on gastric digestion obtained
by Dr. Beaumont on Alexis St. Martin, followed a little later, in
1842, by a long period of experimentation by many physiologists,
as Blondlot,[3] Bassow,[4] Bardeleben,[5] Bernard,[6] Bidder and
Schmidt,[7] and many others on methods of establishing gastric fistulæ
on animals, by which many interesting results were accumulated
regarding the physiology of gastric digestion. Up to 1834, however,
there was no adequate explanation offered of the solvent power of
the stomach juice; aside from the presence of hydrochloric acid,
nothing could be discovered by the earlier chemists to account for the
remarkable digestive action. Eberle,[8] however, attributed to the
mucous membrane of the stomach a catalytic action, and claimed that it
only needed the presence of a small piece of the stomach mucosa with
weak hydrochloric acid for the manifestation of solvent or digestive
power. It remained for Schwann,[9] to show the true explanation of this
phenomenon, and although he was unable to make a complete separation
of the active principle which he plainly believed existed, he gave to
it the name of pepsin. Wassmann, Pappenheim,[10] Valentin, and later
Elsässer,[11] all endeavored to obtain the substance in a pure form,
and Wassmann,[12] in 1839, surely succeeded in obtaining a very active
preparation of the ferment--one capable of exerting marked digestive
action when mixed with a little dilute acid. Thus, a true understanding
of the general nature of gastric juice was finally arrived at, and the
cause of its digestive power was rightfully attributed to the presence
of the ferment pepsin and the dilute acid. Further, the analysis of
human gastric juice made by Berzelius,[13] in 1834, showed that the
secretion contains very little solid matter (1.26 per cent.), thus
calling attention to the fact that the digestive power of this fluid is
out of all proportion to the amount of pepsin, or even to the amount
of total solid matter present, and consequently paving the way for
a general appreciation of the peculiar nature of the dominant body,
_i.e._, the pepsin.

[3] Traité analytique de la Digestion. Paris, 1842.

[4] Bulletin de la Société des Naturalistes de Moscou, vol. 16. 1842.

[5] Archiv für physiol. Heilkunde, vol. 8. 1849.

[6] Lecons de Physiologie de la Digestion. Paris, 1867.

[7] Die Verdauungssäfte.

[8] Physiologie der Verdauung. Würzburg, 1834.

[9] Ueber das Wesen der Verdauungsprocesse. Müller’s Archiv, 1836, p.
90.

[10] Zur Kenntniss d. Verdanuung. Breslau, 1839.

[11] Magenerweichung der Säuglinge. Stuttgart und Tübingen, 1846.

[12] Lehmann’s Lehrbuch d. physiol. Chem., Band 2, p. 41, 2te Auflage.

[13] Lehrbuch der Chemie, Band 9, p. 209.

The original conception regarding the manner in which gastric juice
exerts its solvent power on proteid foods was apparently limited to
simple solution; chemical solution if you choose, brought about by
catalytic action, but without any hint as to the possible nature of
the soluble products formed. Mialhe,[14] however, recognized the fact
that this transformation, by which insoluble and non-diffusible proteid
matter was converted into a soluble and diffusible product, was a form
of hydration, comparable to the change of insoluble starch into soluble
sugar, and he named the hypothetical product albuminose. Mialhe’s study
of the matter in 1846 was followed by Lehmann’s[15] investigation of
the subject, and the coining of the word peptones as an appropriate
name for the soluble products of gastric digestion. The peptones
isolated by Lehmann were described as amorphous, tasteless substances,
soluble in water in all proportions and insoluble in alcohol. They
were likewise precipitated by tannic acid, mercuric chloride, and lead
acetate, and were considered as weak acid bodies, having the power
of combining with bases to form salts of a more or less indefinite
character. Twelve years later, in 1858, Mulder[16] gave a more complete
description of peptones, but his study of the subject failed to advance
materially our knowledge of the broader questions regarding the nature
of the process, or processes, by which the so-called peptones were
formed. A year later, in 1859, Meissner[17] brought forward the first
of his contributions, and during the following three or four years
several communications were made representing the work of himself and
pupils upon the question of gastric digestion, or more especially upon
the character of the products resulting from the digestive action of
pepsin-hydrochloric acid.

[14] Canstatt’s Jahresbericht d. Pharm., 1846, p. 163.

[15] Lehmann’s Physiologische Chemie, Band 2, p. 318.

[16] Archiv f. d. Holländ. Beitr., 2, 1858.

[17] Zeitschr. f. rat. Med., Band 7, 8, 10 und 14.

The general tenor of Meissner’s results is shown in the description
of a row of products as characteristic of the proteolytic action of
pepsin-acid on proteid matter. In other words, there was a clear
recognition of the fact that proteid digestion in the stomach, through
the agency of the ferment pepsin, is something more than a simple
conversion of the proteid into one or two soluble products. The several
bodies then isolated were named parapeptone, metapeptone, dyspeptone,
α, β, and γ peptone; names now seldom used, but significant as showing
that at this early date there was a full appreciation of the fact
that digestive proteolysis as accomplished by the ferment pepsin is an
intricate process, accompanied by the formation of a series of products
which vary more or less with the conditions under which the digestion
is conducted.

This was the commencement of our more modern ideas regarding digestive
proteolysis, but only the commencement, for it ushered in an era of
unparalleled activity, in which Brücke, Schützenberger, and Kühne each
contributed a large share toward the successful interpretation of the
results obtained. Further, knowledge regarding the proteid-digesting
power of the pancreatic juice was rapidly accumulating, thus
broadening our ideas regarding digestive proteolysis in general.
Corvisart[18] had called attention to the proteolytic power of the
pancreatic juice in 1857, and although his observations were more or
less generally discredited for a time, they were eventually confirmed
by Meissner,[19] Schiff, Danilewsky,[20] and Kühne,[21] the latter
particularly contributing greatly to the development of our knowledge
concerning this phase of digestive proteolysis. The proteolytic power
was proved to be due to a specific ferment or enzyme, now universally
called trypsin, which digests proteid foods to the best advantage in
the presence of sodium carbonate. Digestive proteolysis in the human
body was thus shown to be due mainly to the presence of two distinct
enzymes, the one active in an acid fluid, the gastric juice, the other
in an alkaline-reacting fluid, the pancreatic juice, but both endowed
with the power of digesting all varieties of proteid foods, with the
formation of a large number of more or less closely related products.

[18] Sur une Fonction peu connue du Pancréas: La digestion des aliments
azotés. Paris, 1857-58.

[19] Verdauung der Eiweisskörper durch den pankreatischen Saft.
Zeitschr. f. rat. Med., 3d ser., Band 7, p. 17. 1859.

[20] Virchow’s Archiv, Band 25, p. 267. 1862.

[21] _Ibid._, Band 39, p. 130. 1867.

So much for the early history of our subject, and now, without
attempting any exhaustive sketch of its gradual development during
the last decade and a half, allow me to present to you digestive
proteolysis as it stands to-day, developed somewhat, I trust, by the
results I have been able to contribute to it during the last twelve
years.


THE GENERAL NATURE OF PROTEOLYTIC ENZYMES.

These peculiar bodies owe their origin to the constructive power of
the gland-cells from which the respective secretions are derived.
During fasting, the epithelial cells of the gastric glands and of
the pancreas manufacture from the cell-protoplasm a specific zymogen
or ferment-antecedent, which is stored up in the cell in the form
of granules. These granules of either pepsinogen or trypsinogen,
as the case may be, are during secretion apparently drawn upon for
the production of the ferment, and it is an easy matter to verify
Langley’s[22] observation that the amount of pepsin, for example,
obtainable from a definite weight of the gland-bearing mucous
membrane is proportionate to the number of granules contained in
the gland-cells. During ordinary secretion, however, these granules
of zymogen do not entirely disappear from the cell. When secretion
commences and the granules are drawn upon for the production of
ferment, fresh granules are formed, and inasmuch as these latter are
produced through the katabolism of the cell-protoplasm it follows
that anabolic processes must be simultaneously going on in the cell,
by which new cell-protoplasm is constructed. Hence, as Heidenhain,
Langley, and others have pointed out, during digestion there are
at least three distinct processes going on side by side in the
gland-cell, viz., the conversion of the zymogen stored up in the cell
into the active ferment, or other secretory products, the growth of
new cell-protoplasm, and the attendant formation of fresh zymogen to
replace, or partially replace, that used up in the production of the
ferment. Consequently, we are to understand that in the living mucous
membrane of the stomach there is little or no preformed pepsin present.
Similarly, the cells of the pancreatic gland are practically free
from the ferment trypsin. In both cases the cell-protoplasm stores up
zymogen and not the active ferment, but at the moment of secretion
the zymogen is transformed into ferment and possibly other organic
substances characteristic of the fluid secreted. Absorption of the
products of digestion tends to increase the activity of the secreting
cells, but we have no tangible proof that any particular kinds of food
are directly peptogenous, _i.e._, that they lead to a storing up in
the gastric cells, for example, of pepsinogen, although it may be that
the so-called peptogenous foods give rise to a more active conversion
of pepsinogen into pepsin.[23] As already stated, the zymogen is
manufactured directly from the cell-protoplasm, and the constructive
power is certainly not directly controlled by the character of the food
ingested.

[22] Proceedings of the Royal Society, vol. 32, p. 20; On the Histology
and Physiology of the Pepsin-forming Glands.

[23] Langley and Edkins: Pepsinogen and Pepsin. Journal of Physiology,
vol. 7, p. 394.

All this in one sense is to-day ancient history, but I recall it to
your minds in order to emphasize the fact that these two energetic
ferments or enzymes stand in close relation to the protoplasm of
the cell from which they originate. So far as we can measure the
transformations involved, there are only two distinct steps in the
process, viz., the formation of the inactive zymogen stored up in the
cell, and the conversion of the antecedent body into the soluble and
active ferment. In this connection Pod-wyssozki[24] has reported that
the mucous membrane of the stomach exposed to the action of oxygen gas
shows a marked increase in the amount of pepsin, from which he infers
that the natural conversion of pepsinogen into pepsin is an oxidation
process. Further, he claims the existence of at least two forms of
pepsinogen in the stomach mucosa, one closely akin to the ferment
itself and very easily soluble in glycerin, while the other is more
insoluble in this menstruum. Langley and Edkins,[25] however, find
that oxygen has no effect whatever on the pepsinogen of the frog’s
mucous membrane, thus throwing doubt on the above conclusion. Still,
Podolinski[26] claims that trypsin originates from its particular
zymogen through a process of oxidation, and Herzen[27] has proved
that the ferment can be reconverted into trypsinogen under the
influence of carbon-monoxide and again transformed into the ferment by
contact with oxygen gas. This latter observer[28] has also noticed a
connection between the amount of trypsin obtainable from the pancreas
and the dilatation of the spleen, from which he was eventually led
to conclude that the spleen during its dilatation gives birth to a
zymogen-transforming ferment which thus leads to the production of
trypsin, presumably from the already manufactured zymogen. In any
event, their peculiar origin lends favor to the view that these two
enzymes are closely allied to proteid bodies, and that they are
directly derived from the albuminous portion of the cell-protoplasm.
Analysis shows that they always contain nitrogen in fairly large
amount, although the percentage is sometimes less than that found in a
typical proteid body.

[24] Pflüger’s Archiv f. Physiol., Band 39, p. 68.

[25] Journal of Physiol., vol. 7, p. 400.

[26] Pflüger’s Archiv f. Physiol., Band 13, p. 422.

[27] Ueber den Rückschlag des Trypsins zu Zymogen unter dem Einfluss
der Kohlenoxydvergiftungen. Pflüger’s Archiv f. Physiol., Band 30, p.
308.

[28] Ueber den Einfluss der Milz auf die Bildung des Trypsins.
Pflüger’s Archiv f. Physiol., Band 30, p. 295.

It must be remembered, however, that in spite of oft-repeated attempts
to obtain more definite knowledge regarding the composition of these
proteolytic enzymes our efforts have been more or less baffled. We
are confronted at the outset with the fact that no criterion of
chemical purity exists, either in the way of chemical composition
or of chemical reactions. The only standard of purity available is
the intensity of proteolytic action, but this is so dependent upon
attendant circumstances that it is only partially helpful in forming an
estimate of chemical purity. My own experiments in this direction, and
they have been quite numerous, have convinced me that it is practically
impossible to obtain a preparation of either pepsin or trypsin at
all active which does not show at least some proteid reactions.
Furthermore, such samples of these two enzymes as I have analyzed have
shown a composition closely akin to that of proteid bodies. I will not
take time to go into all the details of my work in this direction,
contenting myself here with the statement that the purest specimens of
pepsin and trypsin I have been able to prepare have always shown their
relationship to the proteid bodies by responding to many of the typical
proteid reactions, and their composition, though somewhat variable, has
in the main substantiated this evident relationship.

The most satisfactory method I have found for obtaining a comparatively
pure preparation of pepsin, and one at the same time strongly active,
is a modification of the method published some years ago by Kühne and
myself.[29] The mucous membrane from the cardiac portion of a pig’s
stomach is dissected off and washed with water. The upper surface of
the mucosa is then scraped with a knife until at least half of the
membrane is removed. These scrapings, containing the fragments of the
peptic glands, are warmed at 40° C. with an abundance of 0.2 per cent.
hydrochloric acid for ten to twelve days in order to transform all of
the convertible albuminous matter into peptone. The solution is then
freed from insoluble matter by filtration and immediately saturated
with ammonium sulphate, by which the pepsin, with some albumose, is
precipitated in the form of a more or less gummy, or semi-adherent
mass. This is filtered off, washed with a saturated solution of
ammonium sulphate and then dissolved in 0.2 per cent. hydrochloric
acid. The resultant solution is next dialyzed in running water until
the ammonium salt is entirely removed, thymol being added to prevent
putrefaction, after which the fluid is mixed with an equal volume of
0.4 per cent. hydrochloric acid and again warmed at 40° C. for several
days. The ferment is then once more precipitated by saturation of the
fluid with ammonium sulphate, the precipitate strained off, dissolved
in 0.2 per cent. acid and again dialyzed in running water until the
solution is entirely free from sulphate. The clear solution of the
ferment obtained in this manner can then be concentrated at 40° C. in
shallow dishes, and if desired the ferment obtained as a scaly residue.
So prepared, the pepsin is certainly quite pure, that is comparatively,
and although it may contain some albumose, the latter must be very
resistant to the action of the ferment; indeed, pepsin is in many
respects an albumose-like body itself.

[29] Zeitschr. f. Biol., Band 22, p. 428.

In any event, the enzyme prepared in this manner shows decided proteid
reactions, and contains nitrogen corresponding more or less closely to
the recognized composition of an albumose. My own belief, therefore,
is that these enzymes, both pepsin and trypsin, are proteid bodies
closely related to the albumoses. They are soluble in water and more
or less soluble in glycerin; at least glycerin will dissolve them from
moist tissues, or from moist precipitates containing them. Langley,[30]
however, states, and perhaps justly, that we have no positive proof
that either ferments or zymogens are soluble in pure strong glycerin,
and that if they are soluble, it is extremely slowly. In dilute
glycerin, however, these ferments dissolve readily, as we very well
know. Furthermore, they are practically non-diffusible, and, like many
albumoses, are precipitated in part by saturation with sodium chloride
and completely on saturation with ammonium sulphate.

[30] Gamgee’s Physiological Chemistry of the Animal Body, vol. 2, p. 4.
1893.

When dissolved in water and heated above 80° C., these enzymes are
decomposed to such an extent that their proteolytic power is totally
destroyed. The amount of coagulum produced by heat, however, is
comparatively small, though variable with different preparations. Thus
with trypsin, Kühne originally considered that boiling an aqueous
solution of the ferment would give rise to about twenty per cent.
of coagulated proteid and eighty per cent. of peptone-like matter.
With the purer preparations now obtainable there is apparently less
coagulable matter present, and Loew[31] has succeeded in preparing
from the pancreas of the ox a sample of trypsin containing 52.75 per
cent. of carbon and 16.55 per cent. of nitrogen, and yielding only
a small coagulum by heat. Loew considered the ferment to be a true
peptone, but in view of our present knowledge regarding the albumoses,
I think we are justified in assuming it to be an albumose-like body
rather than a true peptone. At the same time it may be well to again
emphasize the fact that our only “means of determining the presence
of an enzyme is that of ascertaining the change which it is able
to bring about in other substances, and since the activity of the
enzymes is extraordinarily great, a minute trace suffices to produce
a marked effect. From this it follows that the purified enzymes
which give distinct proteid reactions might merely consist of very
small quantities of a true non-proteid enzyme, adherent to or mixed
with a residue of inert proteid material.”[32] This quotation gives
expression to a possibility which we certainly cannot ignore, but my
own experiments lead me to believe firmly in the proteid nature of
these two enzymes. Further, we find partial substantiation of this view
in the results obtained by Wurtz[33] in his study of the vegetable
proteolytic ferment papain, and in my own results from the study of the
proteolytic ferment of pineapple juice.[34] Thus, Wurtz prepared from
the juice of Carica papaya an active sample of papain, and found it to
contain on analysis about 16.7 per cent. of nitrogen and 52.5 per cent.
of carbon, while the reactions of the product likewise testified to the
proteid nature of the enzyme. Martin, too, has concluded from his study
of papain that the ferment is at least associated with an albumose.[35]

[31] Ueber die chemische Natur der ungeformten Fermente. Pflüger’s
Archiv f. Physiol., Band 27, p. 203.

[32] Sheridan Lea: Chemical Basis of the Animal Body, p. 55.

[33] Sur la papaine: Contribution à l’histoire des ferments solubles,
Comptes Rendus, Tome 90, p. 1379. _Ibid._, Tome 91, p. 787.

[34] On the Proteolytic Action of Bromelin, the Ferment of Pineapple
Juice. Journal of Physiol., vol. 15, p. 249.

[35] The Nature of Papain, etc. Journal of Physiol., vol. 6, p. 336.

With the proteolytic ferment of pineapple juice my observations have
led me to the following conclusions, viz., that the ferment is at least
associated with a proteid body, more or less completely precipitable
from a neutral solution by saturation with ammonium sulphate, sodium
chloride, and magnesium sulphate. This body is soluble in water,
and consequently is not precipitated by dialysis. It is further
non-coagulable by long contact with strong alcohol, and its aqueous
solution is very incompletely precipitated by heat. Placing it in line
with the known forms of albuminous bodies it is not far removed from
protoalbumose or heteroalbumose, differing, however, from the latter in
that it is soluble in water without the addition of sodium chloride. At
the same time, it fails to show some of the typical albumose reactions,
and verges toward the group of globulins. In any event, it shows many
characteristic proteid reactions, and contains considerable nitrogen,
viz., 10.46 per cent., with 50.7 per cent, of carbon. Consequently, we
may conclude that the chemical reactions and composition of the more
typical proteolytic enzymes, both of animal and vegetable origin, all
favor the view that they are proteid bodies not far removed from the
albuminous matter of the cell-protoplasm.

Further, the very nature of these substances and their mode of action
strengthen the idea that they are not only derived from the albumin
of the cell-protoplasm, but that they are closely related to it. One
cannot fail to be impressed with the resemblance in functional power
between the unformed ferments as a class and cell-protoplasm. To what
can we ascribe the particular functional power of each individual
ferment? Why, for example, does pepsin act on proteid matter only in
the presence of acid, and trypsin to advantage only in the presence
of alkalies? Why does pepsin act only on proteid matter, and ptyalin
only on starch and dextrins? Why does trypsin produce a different set
of soluble products in the digestion of albumin than pepsin does?
Similarly, why is it that the cell-protoplasm of one class of cells
gives rise to one variety of katabolic products, while the protoplasm
of another class of cells, as in a different tissue or organ, manifests
its activity along totally different lines? The answer to both sets of
questions is, I think, to be found in the chemical constitution of
the cell-protoplasm on the one hand, and in the constitution of the
individual enzymes on the other. The varied functional power of the
ferment is a heritage from the cell-protoplasm, and, as I have said, is
suggestive of a close relationship between the enzymes and the living
protoplasm from which they originate. We might, on purely theoretical
grounds, consider that these unformed ferments are isomeric bodies
all derived from different modifications of albumin and with a common
general structure, but with individual differences due to the extent of
the hypothetical polymerization which attends their formation.

Whenever, owing to any cause, the activity of the ferment is
destroyed, as when it is altered by heat, strong acids, or alkalies,
then the death of the ferment is to be attributed to a change in its
constitution; the atoms in the molecule are rearranged, and as a result
the peculiar ferment power is lost forever. The proteolytic power of
these enzymes is therefore bound up in the chemical constitution of
the bodies, and anything which tends to alter the latter immediately
interferes with their proteolytic action. But how shall we explain
the normal action of these peculiar bodies? Intensely active, capable
in themselves of producing changes in large quantities of material
without being destroyed, their mere presence under suitable conditions
being all powerful to produce profound alterations, these enzymes play
a peculiar part. Present in mere traces, they are able to transform
many thousand times their weight of proteid matter into soluble and
diffusible products. All that is essential is their mere presence under
suitable conditions, and strangely enough the causative agent itself
appears to suffer no marked change from the reactions set up between
the other substances.

There are many theories extant to explain this peculiar method of
chemical change, but few of them help us to any real understanding of
the matter. These enzymes are typical catalytic or contact agents,
and by their presence render possible marked changes in the character
of the proteid or albuminoid matter with which they happen to be
in contact. But the conditions under which the contact takes place
exercise an important control over the activity of the ferment.
Temperature, reaction, concentration of the fluid, presence or absence
of various foreign substances, etc., all play a very important part
in regulating and controlling the activity of these two proteolytic
enzymes. In fact, as one looks over the large number of data which
have gradually accumulated bearing upon this point, one is impressed
with the great sensitiveness of these ferments toward even so-called
indifferent substances. Their specific activity appears to hinge
primarily upon the existence of a certain special environment,
alterations of which may be attended with an utter loss of proteolytic
power, or, in some less common cases, with a decided increase in the
rate of digestive action. This constitutes one of the peculiar features
of these proteolytic enzymes; powerful to produce great changes, they
are nevertheless subject to the influence of their surroundings in a
way which testifies to their utter lack of stability. Furthermore, as
you well know, conditions favorable for the action of the one ferment
are absolutely unfavorable for the activity of the other, and indeed
may even lead to its destruction. Thus, while pepsin requires for its
activity the presence of an acid, as 0.2 per cent. HCl, trypsin is
completely destroyed in such a medium. Again, trypsin exhibits its
peculiar proteolytic power in the presence of sodium carbonate, a salt
which has an immediate destructive action upon pepsin. Hence, a medium
which is favorable for the action of the one ferment may be directly
antagonistic to the action of the other.

Another factor of great moment in determining the activity of these
two enzymes is temperature. That which is most favorable for their
action is 38° to 40° C., and any marked deviation from this temperature
is attended by an immediate effect upon the proteolysis. Exposure to
a low temperature simply <DW44>s proteolytic action, doubtless in
the same manner that cold checks or <DW44>s other chemical changes.
There is no destruction of the ferment, even on exposure to extreme
cold, the enzyme being simply inactive for the time being. Exposure
of either pepsin or trypsin to a high temperature, say 80° C., is
quickly followed by a complete loss of proteolytic power, _i.e._,
the ferment is destroyed. It is to be noticed, however, that the
destructive action of heat is greatly modified by the attendant
circumstances. Thus, fairly pure trypsin, dissolved in 0.3 per cent.
sodium carbonate, is completely destroyed on exposure to a temperature
of 50° C. for five to six minutes, while a neutral or slightly acid
solution of the pure enzyme is destroyed in five minutes by exposure
to a temperature of 45° C. On the other hand, the presence of inorganic
salts and the products of digestion, such as albumoses, amphopeptone,
and antipeptone, all tend to protect the trypsin somewhat from the
destructive effects of high temperatures, so that in their presence
the enzyme may be warmed to 60° C. before it shows any diminution in
proteolytic power. Alkaline reaction, combined with the presence of
salts and proteid, viz., just the conditions existent in the natural
pancreatic secretion, constitute the best safeguard against the
destructive action of heat, and under such conditions trypsin may be
warmed to about 60° C. before it begins to suffer harm. But all this
testifies in no uncertain way to the extreme sensitiveness of the
ferment to changes in temperature; a sensitiveness which manifests
itself not only in diminished or retarded proteolytic action, but
terminates in destruction of the ferment when the temperature rises
beyond a certain point.

Similarly, pepsin dissolved in 0.2 per cent. hydrochloric acid feels
the destructive effect of heat when a temperature of 60° C. is reached.
In a neutral solution, on the other hand, destruction of the ferment
may be complete at 55° C. Here, too, peptone <DW44>s very noticeably the
destructive action of heat, especially in an acid solution of pepsin,
so that under such circumstances the ferment may not be affected until
the temperature reaches 70° C. I have tried many experiments along
this line, not only with pepsin and trypsin, but also with many other
ferments. We may briefly summarize, however, all that is necessary for
us to consider here in the statement that the pure isolated ferments
are far more sensitive to the destructive action of heat than when
they are present in their natural secretions. This, as stated, is due
not only to the reaction of the respective fluids but also to the
protective or inhibitory action of the inorganic salts and various
proteids naturally present. We may thus say with Biernacki[36] that the
purer the ferment the less resistant it is to the effects of heat.

[36] Das Verhalten der Verdauungsenzyme bei Temperaturerhöhungen.
Zeitschr. f. Biol., Band 28, p. 49.

It is thus plain that these enzymes, capable though they are of
accomplishing great tasks, are nevertheless exceedingly unstable and
prone to lose their proteolytic power under the slightest provocation.
When, however, they are surrounded by their natural environment,
the acid or alkali of the respective secretion, together with salts
and proteids, they then appear more stable; their natural lability
becomes for the time being transformed into semi-stability, and the
temperature, for example, at which they lose their peculiar power, is
raised ten degrees or more. I have also found the same to be true of
the vegetable proteolytic ferments, and also of the amylolytic ferment
of saliva.

The above facts furnish us, I think, a good illustration of how
dependent these proteolytic enzymes are upon the proper conditions of
temperature, to say nothing of other conditions, for the full exercise
of their peculiar power. Toward acids, alkalies, metallic salts, and
many other compounds they are even more sensitive than toward heat,
and much might be said regarding the effects, inhibitory or otherwise,
produced by a large number of common drugs or medicinal agents on
these two ferments. Any lengthy discussion of this matter, however,
would be foreign to our subject, and I will only call your attention
in passing to one or two points which have a special bearing upon the
general nature of the enzymes. Take, for example, the influence of
such substances as urethan, paraldehyde, and thallin sulphate on the
proteolytic action of pepsin-hydrochloric acid[37] and we find that
small quantities, 0.1 to 0.3 per cent. tend to increase the rate of
proteolysis, while larger amounts, say one per cent., decidedly check
proteolysis. Similarly, among inorganic compounds, arsenious oxide,
arsenic oxide, boracic acid, and potassium bromide[38] in small amounts
increase the proteolytic power of pepsin in hydrochloric acid solution,
while larger quantities check the action of the ferment in proportion
to the amounts added. Again, with the enzyme trypsin, similar results
with such salts as potassium cyanide, sodium tetraborate, potassium
bromide and iodide[39] may be quoted as showing not only the
sensitiveness of the ferment toward foreign substances, but likewise
its peculiar behavior, viz., stimulation in the presence of small
amounts and inhibition in the presence of larger quantities.

[37] Chittenden and Stewart: Studies in Physiol. Chem., Yale
University. Vol. 3, p. 64.

[38] Chittenden and Allen, _Ibid._, vol. 1, p. 76.

[39] Chittenden and Cummins, _Ibid._, vol. 1, p. 112.

Furthermore, we have found that even gases, as carbonic acid and
hydrogen sulphide, exert a marked retarding influence on the
proteid-digesting power of trypsin. Moreover, while it is generally
stated that proteolytic and other enzymes are practically indifferent
to the presence of chloroform, thymol, and other like substances
that quickly interfere with the processes of the so-called organized
ferments, pepsin and trypsin certainly do show a certain degree of
sensitiveness to chloroform, and indeed even to a current of air
passed through their solutions. Thus, very recently, Bertels[40] and
Dubs[41], working under Salkowski’s direction, have called attention
to the peculiar behavior of pepsin to chloroform; their results
showing, first, that small amounts of this agent tend to increase the
proteolytic power of the enzyme, while larger amounts decrease its
digestive power. Another interesting point brought out especially
by Dub’s experiments is the fact that an impure solution of the
ferment, viz., an acid extract, for example, of the mucous membrane
of the stomach containing more or less albuminous matter, is far less
sensitive to chloroform than an acid solution of the purified ferment,
thus showing again the protective influence of proteids and other
extraneous matters; the latter guarding the enzyme to a certain extent
from both the stimulating and inhibitory action of various agents.

[40] Ueber den Einfluss des Chloroforms auf die Pepsinverdauung,
Virchow’s Archiv, Band 130, p. 497.

[41] Der Einfluss des Chloroforms auf die künstliche Pepsinverdauung,
Ibid., Band 134, p. 519.

Another point to be emphasized just here is that any chemical
substance, such as a metallic salt, having a specific action upon
proteid matter, will almost invariably interfere more or less with
the proteolytic action of these enzymes, both through a direct
action upon the soluble ferment itself, and also through an indirect
action in modifying or inhibiting the digestibility of the proteid
exposed to proteolysis. All of these facts emphasize more or less the
proteid-like nature of the enzymes, or at least the carriers of the
ferments. It is further very suggestive that the destruction of these
enzymes by heat happens to occur at approximately those temperatures
which are generally recognized as the coagulation points of ordinary
proteids. Moreover, the apparent lack of stability so characteristic of
these ferments, their inherent proneness to alteration, their marked
susceptibility to every change in environment, all point to large
complex molecules, such as we have in proteids and are familiar with in
living protoplasm.

Whatever the exact nature of these proteolytic enzymes, they are
certainly endowed with the power of transforming relatively large
amounts of proteid matter into soluble products, even though they
themselves are present in very small quantity. They are derived, as
we have seen, from living protoplasmic cells, and we might perhaps,
with v. Nägeli[42] and Mayer,[43] consider them as retaining a portion
of that molecular motion so characteristic of living protoplasm, by
which the equilibrium of the dead food-proteid may be disturbed and
thus changes started which result in what we call proteolysis. However
this may be, we must look to some phase of catalytic or contact
action as the true explanation of this power of proteolysis. At first
glance, any explanation or theory involving the use of catalysis seems
exceedingly vague and indefinite, and yet many illustrations can be
given of chemical reactions where the dominating agent evidently acts
in this manner. “We call a force catalytic,” says the philosopher of
Heilbron, “when it holds no communicable proportion to the assumed
results of its action. An avalanche is hurled into the valley....
A puff of wind or the fluttering of a bird’s wing is the catalytic
force which has given the signal for, and which is the cause of the
wide-spread disaster.”[44] In the older theories of catalytic action,
the catalytic agent was supposed to remain passive, but not so in the
more modern conception of catalysis. The ferment by its presence makes
possible certain changes and combinations which could not occur in its
absence, at least under the existing circumstances, although all the
other conditions might be favorable. The proteids, for example, have
a natural tendency to undergo hydration; thus, simple boiling with
dilute acid or exposure to the action of superheated water alone,[45]
will produce many if not all of the products formed in natural
digestive proteolysis. To be sure, they are not formed as readily as
in artificial or natural digestion, and there may be some minor points
of difference, but still proteolysis can be imitated in this manner.
The proteolytic enzymes simply help on this natural tendency of proteid
bodies to undergo hydration, and by their presence and action enable
it to occur at lower temperatures than it otherwise could, and at the
same time render it more rapid and complete. This is not accomplished,
however, by simple contact. The enzymes, we may assume, combine in
some manner with the proteid undergoing digestion, starting thereby a
train of reactions in which the proteid and the water present are the
main actors, they being, however, perfectly passive in the absence of
the inciting agent, the enzyme. As expressed by Ganger, “the ferment
phenomena resemble those in which there is apparently a periodic
synthesis and dissociation of the catalyzing agent, which acts in a
similar manner to the agent which explodes a train of gunpowder.”

[42] Theorie der Gährung. München, 1879.

[43] Die Lehre von den chem. Fermenten. Heidelberg, 1882.

[44] Quoted from Gamgee’s Physiological Chemistry of the Animal Body,
vol. 2, p. 7.

[45] Chittenden and Meara. A study of the primary products resulting
from the action of superheated water on coagulated egg-albumin. Journal
of Physiol., vol. 15, p. 501.

We can find many illustrations among chemical phenomena where one
body, even though present in small quantity, acts as a go-between and
makes possible an almost indefinite exchange of matter and energy.
Take as an illustration, the part played by water in determining the
explosion of oxygen and carbon-monoxide gas. Some years ago, Dixon[46]
called attention to the fact that a mixture of these two gases when
perfectly dry would not explode even by contact with red hot platinum
wire. The presence, however, of a small amount of aqueous vapor
would at once cause an explosion to occur. In confirmation of this
observation, Traube[47] has reported that a flame of carbon-monoxide
gas introduced into a perfectly dry atmosphere is at once extinguished.
In a moist atmosphere, on the other hand, the flame will continue to
burn indefinitely, that is as long as the CO gas is supplied. In these
cases, the water, which is so necessary for the appearance of the
reaction, and which furnishes a striking illustration of the action of
a contact or catalytic substance is not purely passive. To be sure,
only a minimal amount is necessary for the combustion of an indefinite
amount of carbonic oxide, but the water enters into the reaction
itself. It is to be noticed that carbonic oxide and water alone, even
at high temperatures, will not react, but in the presence of oxygen
the water is decomposed with formation of hydrogen-peroxide, thus:

    CO + 2 H_{2}O + O_{2} = CO(OH)_{2} + H_{2}O_{2}.

[46] Chemical News, vol. 46, p. 151.

[47] Bericht. d. Deutsch. chem. Gesellsch., Band 18, p. 1890.

The hydrogen-peroxide thus formed combines with carbonic oxide to form
carbonic acid, which in turn is decomposed into the anhydride CO_{2},
with regeneration of water, the latter being available for further
action of the same order:

    H_{2}O_{2} + CO = CO(OH)_{2}

    2 CO(OH)_{2} = 2 CO_{2} + 2 H_{2}O.

Indeed, as can be readily seen from the equations, this may be kept
up indefinitely, a small amount of water, _i.e._, the go-between,
the catalytic agent, sufficing to accomplish the transformation of
almost any amount of carbon-monoxide. This, I think, furnishes an
excellent illustration of the way in which catalytic agents, such as
the proteolytic enzymes, may be supposed to act. It is truly contact
action, but the agent is not purely passive; the enzyme combines with
the substance undergoing proteolysis, and the resultant compound thus
formed is enabled now to combine with water and undergo hydrolysis,
something which could not be accomplished by the proteid and water
alone, that is at body temperature. This new and more complex compound
is naturally less stable and soon undergoes dissociation or cleavage
with a splitting off of the original enzyme for one product, which is
thus available for further action of the same order; while, as other
products, we find the hydrated and otherwise altered substances coming
from the proteid, and whose formation is the ultimate object of the
whole process.

The parallelism between this hypothetical action of the proteolytic
enzymes and the known reactions in the above combustion of carbonic
oxide is certainly very close, and leaves little doubt that this
explanation of enzyme action is, in a general way at least, correct.
Thus the carbonic oxide, CO, brought in contact with pure, dry oxygen
gas (apparently all that is necessary for its direct oxidation into
carbonic acid, CO_{2}), undergoes no change; the burning CO gas is at
once extinguished. Evidently, something more is necessary in order to
start the process of oxidation. So, too, in proteolysis; the process,
as we shall see later on, is essentially one of hydration, but bring
the proteid and the water, or acid-water, together and although all
the conditions are apparently favorable for hydration there is, as
you know, little or no change. But introduce the catalytic agent and
immediately the reaction commences. In the case of the burning CO
gas in contact with oxygen, the water acting as contact agent makes
oxidation possible, enabling the main actors in the transformation
to react upon each other. But, as we have seen, the contact agent is
something more than a mere looker-on, it becomes for the time being an
integral part of the molecule, undergoing change, combining with it and
thus making possible the subsequent alterations characteristic of the
specific transformation, in which, however, the regeneration of the
contact agent is a prominent feature. So, too, with the proteolytic
enzymes, pepsin and trypsin, they are the go-betweens, making possible
the union of the proteids with water by combining with the proteid
molecule and thus paving the way for both hydration and cleavage. In
the cleavage of the complex molecule, we have the regeneration of the
ferment as a prominent feature, and in proteolysis we understand that
the regenerated ferment may act not only upon more of the original
proteid, but likewise upon the primary products of its action, thus
giving rise eventually to a row of more or less closely related
cleavage products. Finally, we can conceive that the enzyme may
gradually be affected by the process, that its regeneration may become
less complete, and thus digestive power be eventually diminished.

Much more might be said in support of the above hypothesis. On the
other hand, some objections might be raised against it, but I know
of no more reasonable explanation of enzyme action than that here
presented, or one which so well accords with all of the known facts
concerning the conditions which modify proteolytic action.[48] Thus,
the influence of heat, of the products of proteolysis, of acids,
alkalies, and various organic and inorganic salts on the action of
these digestive enzymes is such as lends favor to the above view rather
than opposes it.

[48] Compare L. de Jager, Erklärungsversuch über die Wirkungsart
der ungeformten Fermente, Virchow’s Archiv, Band 121, p. 182. Also
Chandelon: Bulletin de l’Academie Royale de Méd. de Belgique, 1887, 1,
p. 289.


THE GENERAL NATURE OF PROTEIDS.

Proteids are confessedly among the most complex bodies the physiologist
has to deal with, while at the same time they are perhaps the most
important, not only in view of their wide-spread distribution through
animal and vegetable tissues, but because of the prominent part they
take in the nutrition of the body. The more our knowledge is broadened
concerning these varied substances, the more we are impressed with
their complexity, and at the same time with the necessity for a more
accurate study of both their composition and constitution. Concerning
the latter, full fruition of our hopes is probably in the distant
future, but every step of advance in this direction adds greatly
to our resources in the interpretation of the varied and complex
changes characteristic of proteid metabolism. Every study of proteid
decomposition adds something to our store of knowledge, and gives
perhaps an added fact available for broadening our deductions.[49]
Moreover, the composition and general reactions of the proteids
may be investigated with full confidence of obtaining many useful
results, which must necessarily be an aid in interpreting the changes
accompanying digestive proteolysis.

[49] See Drechsel: Der Abbau der Eiweisstoffe. Du Bois Reymond’s Archiv
f. Physiol., 1891, p. 248.

Take, for example, the single question of peptonization by gastric
digestion. What is the nature of the process? Is the proteid
transformed into a soluble and diffusible peptone as a result of
hydration and cleavage, or is it a transformation which results from
a simple depolymerization of the proteid molecule, _i.e._, are we to
consider albumin and peptone as isomeric bodies? These questions, on
which physiologists seem loath to agree, can certainly be answered
definitely; not, however, by arguments but by careful experimentation,
in which the composition of the proteid undergoing digestion must be
a necessary preliminary factor, and the composition of the resultant
product, or products, a secondary factor of equal importance. Further,
the question needs to be answered not with reference to one proteid
merely, but with reference to every proteid capable of digestion by
either gastric or pancreatic juice. When these questions have been
fully answered in this manner, we shall have positive data to deal
with, and our conclusions will rest upon a foundation of fact not
easily set aside. This is one of the problems upon which I have been at
work for some years, and although progress may in one sense be slow,
yet it is sure and gives results of no uncertain character.

First, then, let us consider briefly the nature of the proteids whose
proteolysis we may be interested in; remembering, however, that in so
doing we can merely touch upon the points essential for our purpose.
Allow me to say in parenthesis that there is being published in Moscow
a work on proteids alone of five volumes, 900 pages each, which it is
supposed will constitute an exhaustive treatise of the subject.[50]

[50] Die Einheit der Proteinstoffe. Historische u. experimentelle
Untersuchungen, by L. Morokhowetz.

If we attempt to classify all of the proteid bodies hitherto discovered
and studied we are at once confronted with a problem of no small
proportions. So varied are they in their reactions, solubilities, and
behavior toward general reagents, so inclined to merge into each other
by almost insensible gradations that it becomes an extremely difficult
matter to make an arrangement that will satisfy all the requirements
of the case. I have to suggest, however, the following classification,
which is merely a modification of several existing ones, based
primarily upon chemical composition, and solubility in the more common
menstruums.

Proteids may first be divided into three main groups as follows:

I. _Simple Proteids._--Composed of carbon, hydrogen, nitrogen, sulphur,
and oxygen, and yielding by decomposition aromatic bodies such as
tyrosin, phenol, indol, etc.

II. _Compound Proteids._--Composed of a simple proteid united to some
non-proteid body.

III. _Albuminoids._--A class of nitrogenous bodies related to and
derived from proteids, but differing especially from the latter by
great resistance to the ordinary solvents of true proteids.

The individual members of these three groups may be arranged as follows
on the basis of solubility, coagulability, etc.:

I. SIMPLE PROTEIDS.--_A._ _Soluble in water._--_a._ Coagulable by heat,
and by long contact with alcohol. Albumins: serum-albumin, egg-albumin,
lacto-albumin, myo-albumin, vegetable albumins. _b._ Non-coagulable by
heat and by long contact with alcohol. Proteoses:[51] protoproteoses,
deuteroproteoses. Peptones:[51] amphopeptones, antipeptones,
hemipeptones.

[51] Used in the generic sense; the proteoses including albumoses,
globuloses, myosinoses, elastoses, etc., and the peptones the
peptone-products formed in the digestion of any or all proteids.

_B._ _Insoluble in water, but soluble in salt solutions._--_a._ More or
less coagulable by heat. Globulins. 1. Soluble in dilute and saturated
NaCl solutions. Vitellins. 2. Soluble in dilute NaCl solutions, but
precipitated by saturation with NaCl. Myosins, paraglobulin[52]
or serum-globulin, fibrinogen, myo-globulin, paramyosinogen,
cell-globulins. _b._ Non-coagulable by heat, soluble in dilute NaCl
solution and precipitated by saturation with NaCl. Heteroproteoses.

[52] Not completely insoluble in saturated NaCl solution.

_C._ _Insoluble in water and salt solutions, soluble in dilute
alcohol_--Zein, gliadins.

_D._ _Insoluble in water, salt solutions and alcohol; soluble in
dilute acids or alkalies._--_a._ Coagulable by heat when suspended in
a neutral fluid. Acid-albumins, alkali-albumins or albuminates. _b._
Non-coagulable by heat when suspended in a neutral fluid. Antialbumids,
dysproteoses, glutenins.

_E._ _Insoluble in water, salt solutions, alcohol, dilute acids and
alkalies; soluble in strong acids, alkalies, and in pepsin-hydrochloric
acid and alkaline solutions of trypsin._--Coagulated proteids,
fibrin.[53]

[53] In blood-fibrin we have a good illustration of the fact that these
divisions are not absolutely exact, since this form of proteid matter,
for example, is somewhat soluble in dilute acids and in salt solutions,
although requiring a long time for marked solution.

II. COMPOUND PROTEIDS.--_A._ _Compounds of a proteid (globulin) with an
iron-containing pigment, soluble in water and coagulable by heat and
alcohol._ Hæmoglobin, oxyhæmoglobin, methæmoglobin, etc.

_B._ _Compounds of proteids with members of the carbohydrate group.
Insoluble in water; soluble in very weak alkalies._--_a._ True mucins.
_b._ Mucoids or mucinoids.

_C._ _Compounds of proteids with nucleic acid. Phosphorized bodies
yielding by decomposition metaphosphoric acid. Insoluble in water
and in pepsin-hydrochloric acid, but more or less soluble in
alkalies._--Nucleins.

_D._ _Compounds of proteids with nucleins. Very soluble in dilute
alkalies._--Nucleoalbumins, as casein of milk, and nucleoalbumins of
cell-protoplasm and cell-nuclei, etc.

III. ALBUMINOIDS.--_A._ _Soluble in boiling water with formation of
gelatin and yielding by decomposition leucin and glycocoll._--Collagen
(gelatin).

_B._ _Insoluble in boiling water, and yielding by decomposition
much leucin and some tyrosin, together with glycocoll and lysatin.
Slowly hydrated by boiling dilute acids and by treatment with
pepsin-hydrochloric acid._--Elastin.

_C._ _Insoluble in water, dilute acids and alkalies, also in
gastric and pancreatic juice. Yield leucin and tyrosin by
decomposition._--Keratin, neurokeratin.

We may now advantageously consider the composition of a few of the
more prominent representatives of the individual groups, taking
for illustration those bodies which have been most thoroughly
studied, and which we may have occasion to refer to in our
discussion of proteolysis. I have not included in the table any of
the alteration-products of the proteids formed by the action of
pepsin-acid, trypsin, or boiling dilute acids, confining myself here
simply to those bodies which occur ready-formed in nature.

COMPOSITION OF SOME OF THE MORE PROMINENT PROTEIDS OCCURRING IN
NATURE.[A]

  ===============================================================================
   Substance. |  C  |  H |  N  |  S |  O  |  P | Ash.|  Origin.  |  Author.
  ------------+-----+----+-----+----+-----+----+-----+-----------+---------------
  Serum-      |53.05|6.85|16.04|1.77|22.29|    |{0.57|Serum from |Hammarsten.[54]
   albumin    |     |    |     |    |     |    |{--  |horse blood|
  Serum-      |52.25|6.65|15.88|2.27|22.95|    |{1.84|Pleural    |Hammarsten.[54]
   albumin    |     |    |     |    |     |    |     |exudation  |
  Egg-albumin |52.25|6.90|15.25|1.93|23.67|    |     |Non-       |Hammarsten.[54]
              |     |    |     |    |     |    |     |coagulated |
  Egg-albumin |52.33|6.98|15.89|1.83|22.97|    | 1.11|Non-       |Chittenden and
              |     |    |     |    |     |    |     |coagulated |Bolton.[55]
  Lacto-      |52.19|7.18|15.77|1.73|23.13|    |     |Cow’s milk |Sebelien.[56]
   albumin    |     |    |     |    |     |    |     |           |
  Vegetable-  |52.25|6.76|16.07|1.48|23.44|    | 0.70|Corn       |Chittenden and
   albumin    |     |    |     |    |     |    |     |or maize   |Osborne.[57]
  Vegetable-  |53.02|6.84|16.80|1.28|22.06|    | 0.82|Wheat      |Osborne and
   albumin    |     |    |     |    |     |    |     |           |Voorhees.[58]
  Proteose,   |52.13|6.83|16.55|1.09|23.40|    | 0.79|Hemialbu-  |Kühne and
   animal     |     |    |     |    |     |    |     |mose, urine|Chittenden.[59]
  Proteose,   |50.60|6.68|16.33|1.62|24.77|    | 2.99|Corn       |Chittenden and
   vegetable  |     |    |     |    |     |    |     |or maize   |Osborne.[57]
  Proteose,   |51.86|6.82|17.32|    |     |    | 0.25|Wheat      |Osborne and
   vegetable  |     |    |     |    |     |    |     |           |Voorhees.[58]
  Proteose,   |49.98|6.95|18.78|    |     |    | 1.80|Flax-seed  |Osborne.[60]
   vegetable  |     |    |     |    |     |    |     |           |
  Proteose,   |46.52|6.40|18.25|    |     |    | 2.20|Cocoanut   |Chittenden and
   vegetable  |     |    |     |    |     |    |     |meat       |Setchell.[61]
  Vitellin,   |51.71|6.84|18.12|0.85|22.48|    | 1.20|Corn       |Chittenden and
   spheroidal |     |    |     |    |     |    |     |or maize   |Osborne.[57]
  Vitellin,   |51.60|6.97|18.80|1.01|21.62|    | 0.30|Squash-seed|Chittenden and
   crystalline|     |    |     |    |     |    |     |           |Hartwell.[62]
  Vitellin,   |51.81|6.94|18.71|1.01|21.53|    |  0  |Squash-seed|Chittenden and
   amorphous  |     |    |     |    |     |    |     |           |Hartwell.[61]
  Vitellin,   |51.48|6.94|18.60|0.81|22.17|    | 0.54|Flax-seed  |Osborne.[60]
   crystalline|     |    |     |    |     |    |     |           |
  Vitellin,   |51.03|6.85|18.39|0.69|23.04|    | 0.49|Wheat      |Osborne and
   spheroids  |     |    |     |    |     |    |     |           |Voorhees.[58]
  Vitellin,   |51.63|6.90|18.78|0.90|21.79|    | 0.56|Hemp-seed  |Chittenden and
   crystalline|     |    |     |    |     |    |     |           |Mendel.[61]
  Vitellin,   |51.31|6.97|18.75|0.76|22.21|    | 0.03|Castor bean|Osborne.[63]
   crystalline|     |    |     |    |     |    |     |           |
  Vitellin,   |52.18|6.92|18.30|1.06|21.54|    | 0.20|Brazil nut |Osborne.[63]
   crystalline|     |    |     |    |     |    |     |           |
  Vitellin,   |51.23|6.90|18.40|1.06|22.41|    | 0.25|Cocoanut   |Chittenden and
   semi-      |     |    |     |    |     |    |     |meat       |Setchell.[62]
   crystalline|     |    |     |    |     |    |     |           |
  Myosin, 13  |52.82|7.11|16.77|1.27|21.90|    | 1.45|Muscle-    |Chittenden and
   different  |     |    |     |    |     |    |     |tissue     |Cummins.[64]
   samples    |     |    |     |    |     |    |     |           |
  Myosin,     |52.68|7.02|16.78|1.30|22.22|    | 0.63|Corn or    |Chittenden and
   vegetable  |     |    |     |    |     |    |     |maize      |Osborne.[57]
  Myosin,     |52.18|7.05|17.90|0.53|22.34|    | 0.10|Oats       |Osborne.[65]
   vegetable, |     |    |     |    |     |    |     |           |
   crystalline|     |    |     |    |     |    |     |           |
  Paraglobulin|52.71|7.01|15.85|1.11|23.24|    | 0.30|Blood      |Hammarsten.[66]
              |     |    |     |    |     |    |     | of horse  |
  Fibrinogen  |52.93|6.90|16.66|1.25|22.26|    | 1.75|Blood      |Hammarsten.[67]
              |     |    |     |    |     |    |     | of horse  |
  Zein        |55.23|7.26|16.13|0.60|20.78|    | 0.43|Corn or    |Chittenden and
              |     |    |     |    |     |    |     |maize      |Osborne.[57]
  Gliadin     |52.72|6.86|17.66|1.14|21.62|    | 0.51|Wheat      |Osborne and
              |     |    |     |    |     |    |     |           |Voorhees.[58]
  Gliadin     |53.01|6.91|16.43|2.26|21.39|    |     |Oats       |Osborne.[63]
              |     |    |     |    |     |    |     |           |
  Glutenin    |52.34|6.83|17.49|1.08|22.25|    |     |Wheat      |Osborne and
              |     |    |     |    |     |    |     |           |Voorhees.[58]
  Coagulated  |52.33|6.98|15.84|1.81|23.04|    | 0.27|Egg-albumin|Chittenden
    proteid   |     |    |     |    |     |    |     |           |and Bolton.[55]
  Coagulated  |51.58|6.88|18.80|1.09|21.65|    | 0.25|Vitellin,  |Chittenden
    proteid   |     |    |     |    |     |    |     |hemp-seed  |and Mendel.[61]
  Fibrin      |52.68|6.83|16.91|1.10|22.48|    | 0.56|Blood      |Hammarsten.[67]
              |     |    |     |    |     |    |     |of horse   |
  Oxyhæmo-    |53.85|7.32|16.17|0.39|21.84|    | 0.43|Blood of   |Hoppe-
   globin     |     |    |     |    |     |    |  Fe.|dog        | Seyler.[68]
  Oxyhæmo-    |54.71|7.38|17.43|0.48|19.60|    | 0.39|Blood of   |Hütner.[69]
   globin     |     |    |     |    |     |    |  Fe.|pig        |
  Mucin       |50.30|6.84|13.62|1.71|27.53|    | 0.33|From snail |Hammarsten.[70]
              |     |    |     |    |     |    |     |           |
  Mucin       |48.84|6.80|12.32|0.84|31.20|    | 0.35|Submaxil-  |Hammarsten.[71]
              |     |    |     |    |     |    |     |liary gland|
  Chondro-    |47.30|6.42|12.58|2.42|31.28|    |     |Cartilage  |Mörner.[72]
   mucoid     |     |    |     |    |     |    |     |           |
  Nuclein     |50.60|7.60|13.18|    |     |1.89|     |Human brain|V. Jaksch.[73]
              |     |    |     |    |     |    |     |           |
  Nuclein     |49.58|7.10|15.02|    |     |2.28|     |Pus        |Hoppe-
              |     |    |     |    |     |    |     |           | Seyler.[74]
  Casein      |52.96|7.05|15.65|0.71|22.78|0.84|     |Cow’s milk |Hammarsten.[75]
              |     |    |     |    |     |    |     |           |
  Casein      |53.30|7.07|15.91|0.82|22.03|0.87| 0.98|Cow’s milk |Chittenden and
              |     |    |     |    |     |    |     |           |Painter.[76]
  Nucleo-     |48.41|7.21|16.85|0.70|24.41|2.42|     |Leucocytes |Lilienfeld.[77]
   histon or  |     |    |     |    |     |    |     |           |
   leuco-     |     |    |     |    |     |    |     |           |
   nuclein    |     |    |     |    |     |    |     |           |
  Gelatin     |49.38|6.81|17.97|0.71|25.13|    | 1.26|Connective |Chittenden and
              |     |    |     |    |     |    |     |tissue     |Solley.[78]
  Elastin     |54.24|7.27|16.70|0.30|21.79|    | 0.90|Neck-band  |Chittenden
              |     |    |     |    |     |    |     |           |and Hart.[79]
  Elastin     |53.95|7.03|16.67|0.38|21.97|    | 0.72|Aorta      |Schwarz.[80]
              |     |    |     |    |     |    |     |           |
  Keratin     |49.45|6.52|16.81|4.02|23.20|    | 1.01|White      |Kühne and
              |     |    |     |    |     |    |     |rabbit’s   |Chittenden.[81]
              |     |    |     |    |     |    |     |hair       |
  Neurokeratin|56.99|7.53|13.15|1.87|20.46|    | 1.35|Human brain|Kühne and
              |     |    |     |    |     |    |     |           |Chittenden.[82]
  Reticulin   |52.88|6.97|15.63|1.88|22.30|0.34| 2.27|Reticular  |Siegfried.[83]
              |     |    |     |    |     |    |     |tissue     |
  ------------+-----+----+-----+----+-----+----+-----+-----------+---------------

[A] Many of these results represent the average of a large number of
individual analyses.

[54] Jahresbericht f. Thierchemie, Band 11, p. 19.

[55] Studies in Physiol. Chemistry, Yale Univer., vol. 2, p. 126.

[56] Zeitschr. physiol. Chem., Band 9, p. 463.

[57] Amer. Chemical Journal, vols. 13 and 14.

[58] Ibid., vol. 15, p. 379.

[59] Zeitschr. f. Biol., Band 19, p. 198.

[60] Amer. Chemical Journal, vol. 14, p. 629.

[61] Not hitherto published.

[62] Journal of Physiology, vol. 11, p. 435.

[63] Amer. Chemical Journal, vol. 14, p. 662.

[64] Studies in Physiol. Chemistry, Yale University, vol. 3, p. 115.

[65] Fourteenth Annual Report Conn. Ag. Exp. Sta., 1890; 2d paper,
Amer. Chemical Journal, vol. 14, p. 212.

[66] Pflüger’s Archiv f. Physiol., Band 22, p. 489.

[67] Ibid., Band 22, p. 479.

[68] Hoppe-Seyler’s Med. Chem. Untersuch, p. 189.

[69] Hoppe-Seyler’s Chem. Analyse, 6th auflage, p. 275.

[70] Pflüger’s Archiv f. Physiol., Band 36. p. 392.

[71] Zeitschr. f. physiol. Chem., Band 12. p. 185.

[72] Jahresbericht f. Thierchemie, Band 18, p. 219.

[73] Pflüger’s Archiv f. Physiol., Band 13, p. 469.

[74] Hoppe-Seyler’s Med. Chem. Untersuch, p. 489.

[75] Zeitschr. f. physiol. Chem., Band 7, p. 269.

[76] Studies in Physiol. Chemistry, Yale University, vol. 2, p. 172.

[77] Du Bois Reymond’s Archiv f. Physiol., 1892, p. 170.

[78] Journal of Physiology, vol. 12. p. 23.

[79] Studies in Physiol. Chemistry, Yale University, vol. 3, p. 19;
also Zeitschr. f. Biol., Band 25, p. 368.

[80] Zeitschr. f. physiol. Chem., Band 18, p. 491.

[81] Zeitschr. f. Biol., Band 26, p. 304.

[82] Ibid., p. 301.

[83] Jahresbericht f. Thierchemie, Band 22, p. 15.

In considering the results tabulated above, it is to be remembered
that all of these bodies, with the exception of keratin, neurokeratin,
and reticulin, are more or less digestible in either gastric or
pancreatic juice, or indeed in both fluids. I will not take time here
to point out the obvious genetic relationships and differences in
composition shown by the above data, but will immediately call your
attention to the fact that there are other and more important points
of difference between many of these proteids which are hidden beneath
the surface, and which a simple determination of composition will not
bring to light. I refer to the chemical constitution of the bodies, to
the way in which the individual atoms are arranged in the molecule,
on which hinges more or less the general properties of the bodies and
which in part determines their behavior toward the digestive enzymes,
as well as toward other hydrolytic agents. These differences in inner
structure can only be ascertained by a study of the decomposition
products of the proteids, and of the way in which the complex molecules
break down into simpler. The nature of the fragments resulting from the
decomposition of a complex proteid molecule, gives at once something
of an insight into the character of the molecule. Thus, egg-albumin
exposed to the action of boiling dilute sulphuric acid yields, among
other fragments, large quantities of leucin and tyrosin, the latter
belonging to the aromatic group and containing the phenyl radical.
Collagen, or gelatin, on the other hand, by similar treatment fails
to yield any tyrosin or related aromatic body, but gives instead
glycocoll or amido-acetic acid, in addition to leucin, lysin, and other
products common to albumin. Its constitution, therefore, is evidently
quite different from that of albumin, but the composition of the body
reveals no sign of it. Further, we have physiological evidence of this
difference in constitution in that gelatin, though containing even
more nitrogen than albumin, is not able to take the place of the latter
in supplying the physiological needs of the body; its food-value is of
quite a different order from that of albumin.

But while all of the individual proteids show many points of
difference, either in composition, constitution, reactions, or
otherwise, they are nearly all alike in their tendency to undergo
hydrolytic decomposition under proper conditions; the extent of the
hydrolysis and accompanying cleavage being dependent simply upon the
vigor or duration of the hydrolytic process.

Furthermore, all of the simple proteids, at least, give evidence of
the presence of two distinct groups or radicals, which give rise by
decomposition or cleavage to two distinct classes of products. These
two groups, which we may assume to be characteristic of every typical
proteid, Kühne has named the anti- and hemi-group respectively. This
conception of the proteid molecule is one of the foundation-stones
on which rest some of our present theories regarding the hydrolytic
decomposition of proteids, especially by the proteolytic enzymes.
Moreover, it is not a mere conception, for it has been tested so
many times by experiment that it has seemingly become a fact. The
two groups, or their representatives, can be separated, in part, at
least, by the action of dilute sulphuric acid (three per cent.) at
100° C. Thus, after a few hours’ treatment of coagulated egg-albumin,
about fifty per cent. of the proteid passes into solution, while there
remains a homogeneous mass, something like silica in appearance,
insoluble in dilute acid, but readily soluble in dilute solutions of
sodium carbonate. This latter is the representative of the anti-group,
originally named by Schützenberger[84] hemiprotein, but now called
antialbumid.[85] It is only slightly digestible in gastric juice, but
is readily attacked by alkaline solutions of trypsin, being converted
thereby into a soluble peptone known as antipeptone. In the sulphuric
acid solution, on the other hand, are found the representatives
of the hemi-group; viz., albumoses, originally known as one body,
hemialbumose,[86] together with more or less hemipeptone, leucin,
tyrosin, etc.

[84] Recherches sur l’albumine et les matières albuminoides. Bulletin
de la Société chimique de Paris, vols. 23 and 24.

[85] Kühne: Weitere Mittheilungen über Verdauungsenzyme und die
Verdauung der Albumine. Verhandl. d. Naturhist. Med. Ver. zu
Heidelberg, Band 1, p. 236.

[86] Kühne und Chittenden: Ueber die nächsten Spaltungsproducte der
Eiweisskörper. Zeitschr. f. Biol., Band 19, p. 159.

The fact that we have so many representatives of the hemi-group in
this decomposition is significant of the readiness with which the
so-called hemi-group undergoes change. All of its members are prone
to suffer hydration and cleavage, passing through successive stages
until leucin, tyrosin, and other simple bodies are reached. These,
and other similar crystalline bodies, are likewise the typical
end-products of proteolysis by trypsin, and presumably come directly
from the breaking-down of hemipeptone. Antipeptone, on the other
hand, is incapable of further change by the proteolytic ferment
trypsin. Hence, the hemi-group can be identified by the behavior of
the body containing it toward trypsin; _i.e._, it will ultimately
yield leucin, tyrosin, and other bodies of simple constitution to be
spoken of later on. The anti-group, however, will show its presence by
a certain degree of resistance to the action of trypsin, antipeptone
being the final product of its transformation by this agent; _i.e._,
leucin, tyrosin, etc., will not result. In this hydrolytic cleavage
of proteids the anti-group does not always appear as antialbumid. It
may make its appearance in the form of some related body, the exact
character of the product being dependent in great part upon the nature
of the hydrolytic agent, but in every case the characteristics of the
anti-group will come to the surface when the body is subjected to the
action of trypsin.

The above-described treatment of a coagulated proteid with water
containing sulphuric acid evidently induces profound changes in the
proteid molecule. The conditions are certainly such as favor hydration,
and in the case of complex molecules, like the proteids, cleavage might
naturally be expected to follow. Analysis of antialbumid from various
sources plainly shows that its formation is accompanied by marked
chemical changes. Thus, the following data, showing the composition of
antialbumid formed from egg-albumin and serum-albumin by the action
of dilute sulphuric acid at 100° C., gives tangible expression to the
extent of this change:

  ===========================================================
          |        |Antialbumid[87]|        | Antialbumid[87]
          |  Egg-  |     from      | Serum- |      from
          |albumin.| egg-albumin.  |albumin.|  serum-albumin.
  --------+--------+---------------+--------+----------------
  C.......| 52.33  |     53.79     | 53.05  |      54.51
  H.......|  6.98  |      7.08     |  6.85  |       7.27
  N.......| 15.84  |     14.55     | 16.04  |      14.31
  --------+--------+---------------+--------+----------------

[87] Kühne und Chittenden: Zeitschr. f. Biol., Band 19, pp. 167 and 178.

In both cases there is a noticeable decrease in nitrogen, and a
corresponding increase in the content of carbon. Evidently, then,
this cleavage of the albumin-molecule into the anti-group on the one
hand, and into bodies of the hemi-group on the other, is accompanied
by chemical changes of such magnitude that their imprint is plainly
visible upon the resultant products; changes which certainly are far
removed from those common to polymerization.

This proneness of proteid matter to undergo hydration and subsequent
cleavage is further testified to by the readiness with which even such
a resistant body as coagulated egg-albumin breaks down under the simple
influence of superheated water at 130° to 150° C. Many observations are
recorded bearing on this tendency of proteid matter, but few observers
have carried their experiments to a satisfactory conclusion. A recent
study of this question in my own laboratory, has given some very
interesting results.[88] Thus, coagulated egg-albumin placed in sealed
tubes with a little distilled water and exposed to a temperature of
150° C. for three to four hours, rapidly dissolves, leaving, however,
an appreciable residue. The solution reacts alkaline, there is a
separation of sulphur, and in the fluid is to be found not albumin, but
two distinct albumose-like bodies, together with some true peptone, and
a small amount of leucin, tyrosin, and presumably other bodies.[89] The
albumose-like bodies are in many ways quite peculiar. In some respects
they resemble the albumoses formed in ordinary digestion; but in others
they show peculiarities which render them quite unique, so that they
merit the specific name of atmidalbumoses, as suggested by Neumeister.
What, however, I wish to call attention to here is the composition of
these albumoses. Prepared from coagulated egg-albumin by the simple
action of heat and water, they show a deviation from the composition of
the mother-proteid, which plainly implies changes of no slight degree.
This is clearly apparent from the following table:

  ==================================================================
    |Coagulated|Atmidalbumose| Atmidalbumose |Deutero- |
    |   egg-   | precipitated|precipitated by| atmid-  |Antialbumid.
    | albumin. |   by NaCl.  | NaCl + acid.  |albumose.|
  --+----------+-------------+---------------+---------+------------
  C |  52.33   |    55.13    |     55.04     |  51.99  |   53.79
  H |   6.98   |     6.93    |      6.89     |   6.60  |    7.08
  N |  15.84   |    14.28    |     14.17     |  13.25  |   14.55
  S |   1.81   |     1.66    |      ---      |   0.98  |    ---
  O |  23.04   |    22.00    |      ---      |  27.18  |    ---
  --+----------+-------------+---------------+---------+------------

[88] Chittenden and Meara: A Study of the Primary Products Resulting
from the Action of Superheated Water on Coagulated Egg-albumin. Journal
of Physiology, vol. 15, p. 501.

[89] Compare Neumeister’s experiments on blood-fibrin. Ueber die
nächste Einwirkung gespannte Wasserdämpfe auf Proteine und über eine
Gruppe eigenthümlicher Eiweisskörper und Albumosen. Zeitschr. f. Biol.,
Band 26, p. 57.

Here we see that two of these primary albumoses formed by the action
of superheated water, like the previously described antialbumid, show
a loss of nitrogen with a marked increase in the content of carbon.
Evidently, they are related to the antialbumid formed by the action
of dilute acid. They are, however, soluble in water, and in many
ways differ from true antialbumid, but there is evidently an inner
relationship. The so-called deuteroatmidalbumose shows a still more
noticeable falling off in nitrogen and sulphur, while the content
of carbon is more closely allied to that of the mother-proteid. The
albumose precipitable by sodium chloride, although different from an
albumid, evidently comes from the anti-group and is a cleavage product
which in turn may undergo further hydration and splitting by continued
treatment. The so-called deutero-body, on the other hand, may well be a
representative of the hemi-group.[90]

[90] Compare Krukenberg, Sitzungsberichte der Jenaischen Gesellschaft
für Medicin, etc. 1886.

It is not my purpose here to enter into details connected with the
action of superheated water on proteids. Such a course would take
us too far from our present subject, but I do wish to emphasize the
fact that even the most resistant of proteids has an innate tendency
to undergo hydration and cleavage, and that even simple heating with
water alone, at a temperature slightly above 100° C., is sufficient to
induce at least partial solution of the proteid. Further, this solvent
action in the case of water and dilute acids, at least, is certainly
associated with marked chemical changes. It is not mere solution, it
is not simply the formation of one soluble body, but solution of the
proteid is accompanied by the appearance of a row of new products,
in which the terminal bodies are crystalline substances of simple
composition. Further, this conclusion does not rest upon the results
obtained from a single proteid, for I have at various times studied
also the primary products formed in the cleavage of casein, elastin,
zein, and other proteids by the action of hot dilute acid, and in all
cases have obtained evidence of the formation of several proteose-like
bodies, as well as of true peptones.

By the action of more powerful hydrolytic agents, such as boiling
hydrochloric acid to which a little stannous chloride has been added
to prevent oxidation, the proteid molecule may be completely broken
down into simple decomposition products, of which leucin, tyrosin,
aspartic acid, glutamic acid, glucoprotein, lysin, and lysatinin are
typical examples.[91] In other words, by this and other methods of
treatment, which we cannot take time to consider, we can easily break
down the albumin-molecule completely into bodies which, as we shall see
later on, are typical end-products of trypsin-proteolysis, and which
are far removed from the original proteid. But, as we have seen, even
the primary bodies formed in the less profound hydrolysis induced by
superheated water, do not show the composition of the mother-proteid.
Hydration and cleavage leave their marks upon the products, and thereby
we know that solution of the proteid is the result of something more
than a mere rearrangement of the atoms in the molecule.

[91] Hlasiwetz und Habermann, Ann. Chem. u. Pharm., Band 169, p. 150.
Also Drechsel, Du Bois-Reymond’s Archiv f. Physiol., 1891, p. 255.

Further, we are to remember that boiling dilute acid and superheated
water tend to produce a cleavage along specific lines; viz., a cleavage
into the anti- and hemi-groups of the molecule, and as representatives
of these groups we may, in the hydration of every native proteid, look
for two distinct rows of closely related substances.

In digestive proteolysis it will be our purpose to show that cleavage
of much the same order occurs, not necessarily resulting, however,
in the formation of identically the same products, but certainly
accompanied with the production of bodies belonging to the hemi- and
anti-groups, although they may be less sharply separated from each
other than in the cleavage with dilute sulphuric acid.

The body originally described as hemialbumose, and identified as a
product of every gastric digestion, is now known to be a mixture of
closely related substances ordinarily spoken of as albumoses,[92]
or generically as proteoses. These are primary products in the
digestion of every form of proteid matter, intermediate between the
mother-proteid and the peptone which results from the further action
of the proteolytic enzymes. Associated with the hemialbumoses are
corresponding antialbumoses, coming from the anti-half of the proteid
molecule, and differing from their neighbors, the hemi-bodies,
mainly in their behavior toward the ferment trypsin. Thus, we have
the counterpart of the many bodies described by Meissner, although
now arranged systematically and on the basis of structural and other
differences not thought of in his day.

[92] Kühne und Chittenden: Ueber Albumosen, Zeitschr. f. Biol., Band
20, p. 11.

By the initial action of pepsin-acid, proteids are first transformed
into acid-albumin or syntonin, then, by the further action of the
ferment, this body is changed into the primary proteoses, proto and
heteroproteose, of each of which there must be two varieties, a hemi
and an anti. These may then undergo further transformation into what
is known as a secondary proteose, viz., deuteroproteose, of which
there must likewise be two varieties, corresponding to the hemi- and
anti-groups respectively. By continued proteolytic action there results
as the final product of gastric digestion peptones; approximately, an
equal mixture of so-called hemipeptone and antipeptone, generally known
as amphopeptone. Such a peptone exposed to the proteolytic action of
trypsin should obviously break down in part into simple crystalline
bodies, leaving a residue of true antipeptone. In truth, this is
exactly what does happen when the peptone resulting from gastric
digestion is warmed with an alkaline solution of trypsin. The so-called
hemipeptone quickly responds to the action of the pancreatic ferment,
and is converted into other products, while the so-called antipeptone
resists its action completely, thus giving results in harmony with our
general conception of the proteid molecule.

ALBUMIN MOLECULE.

    (_Hemi-groups._          _Anti-groups._)
                 ║ ╲       ╱ ║           ║
                 ║   ╲   ╱   ║           ║
                 ║      ╳      ║           ║
                 ║   ╱   ╲   ║           ║
                 ║ ╱       ╲ ║           ║
         Protoalbumose    Heteroalbumose  Antialbumid
        (amphoalbumose)  (amphoalbumose)       ║
               ║  |          |  ║             ║
               ║  |          |  ║             ║
        Deuteroalbumose  Deuteroalbumose Deuteroalbumose
        (amphoalbumose)  (amphoalbumose) (antialbumose)
               ║  |          |  ║             ║
               ║  |          |  ║             ║
          Amphopeptone    Amphopeptone    Antipeptone

On the basis of these facts, and others not yet mentioned, we may
accept provisionally, at least, the above schematic view, suggested
in part by Neumeister,[93] of the general line of proteolysis as
it occurs in pepsin-digestion; a view which clearly expresses the
significant relationship of the hemi- and anti-groups in the proteid
molecule.

[93] Zur Kentniss der Albumosen, Zeitschr. f. Biol., Band 23, p. 391.

The dark and light lines in this scheme are intended to represent the
relative share which the hemi- and anti-groups take in the formation
of the individual bodies. Thus, we see that protoproteoses have their
origin mainly in the hemi-groups of the molecule, although, as the
fine line indicates, anti-groups are somewhat concerned in their
construction. Heteroproteoses, on the other hand, come mainly from
the anti-groups, but still some hemi-groups have a part in their
structure. As previously stated, these two primary proteoses by further
hydrolytic action may be transformed into secondary products; viz.,
into deuteroproteoses, but, as the above scheme indicates, the two
deutero bodies will be more or less unlike in their inner nature. In
one sense, they are both amphodeuteroproteoses, but they necessarily
differ in the proportion of hemi- and anti-groups they contain. By
the still further action of pepsin-acid, the deutero bodies may be
changed, in part at least, into peptone, _i. e._, into amphopeptone,
although, as Neumeister has pointed out, protoproteose tends to yield
an amphopeptone in which the hemi-groups predominate, while the peptone
coming from heteroproteose contains an excess of anti-groups. Moreover,
in the gastric digestion of any simple proteid a certain number of
anti-groups are split off in the form of antialbumid, a body which is
only slowly digestible in pepsin-acid. By the very powerful proteolytic
action of a strong gastric juice, however, antialbumid may be somewhat
digested, and is then transformed into antideuteroalbumose, which in
turn may be eventually changed into antipeptone.

From these statements it is evident that a given proteid exposed to
pepsin-proteolysis may give rise to a large number of products; in
fact, to a far larger number than is implied by the names in the above
scheme. Thus, at first glance you would be inclined to say there can
be only three deuteroalbumoses, for example; one, a pure antibody, the
other two, amphoalbumoses, differing from each other simply in their
content of hemi- and anti-groups. It must be remembered, however, that
the inner constitution of these bodies, as implied by the relative
proportion of the above groups, may vary to almost any extent. Thus,
every variation in the number of anti-groups split off from the
original albumin molecule to form antialbumid means just so much of
a change in the relative proportion of hemi- and anti-groups entering
into the structure of both primary and secondary albumoses. Hence, as
you can see, digestive proteolysis, even in gastric digestion, is a
somewhat complex process. We have to deal not only with a number of
bodies superficially unlike, as the primary and secondary proteoses
and peptones, but these bodies may show marked variations in structure
dependent upon the exact conditions attending their formation.

Evidently, the complexities attending digestive proteolysis are
connected primarily with the complex nature of the proteids themselves,
while proteolysis, as a process, is made possible through the natural
tendency of the proteids to undergo hydration and cleavage.




LECTURE II.

PROTEOLYSIS BY PEPSIN-HYDROCHLORIC ACID, WITH A CONSIDERATION OF THE
GENERAL NATURE OF PROTEOSES AND PEPTONES.


PROTEOLYSIS BY PEPSIN-ACID.

Gastric digestion is essentially an acid digestion. As a proteolytic
agent, pepsin can act only in the presence of acid, and we have every
reason for believing that the enzyme and the acid form a compound,
which in turn combines with the proteid undergoing digestion; or, what
amounts to much the same thing, that the acid perhaps forms first a
compound with the proteid, to which the pepsin can then unite to form
a still more complex compound capable of undergoing hydration and
cleavage. Pepsin-proteolysis, therefore, is strictly the proteolysis
produced by pepsin-acid. In view of this fact, we may well give a
moment’s thought to the nature and origin of this acid.

Without attempting any statement of the gradual development of our
knowledge regarding the acid of the gastric juice, we may accept the
now well-established fact that the acid is hydrochloric acid, and
that it has its origin in the parietal, or so-called border-cells of
the gastric glands. That the acid is derived from the decomposition
of chlorides is practically self-evident, but Cahn[94] has added
experimental proof which removes all shadow of doubt, through his study
of the gastric secretion in animals deprived for many days of salt; the
gastric juice in such cases being perfectly neutral in reaction, but
normal as regards its content of pepsin.

[94] Die Magenverdauung im chlorhunger. Zeitschr. f. physiol. Chem.,
Band 10, p. 522.

The way in which the specific gland-cells manufacture free hydrochloric
acid out of material contained in an alkaline medium is somewhat
doubtful. There are, however, at the present day two theories worthy of
special notice. The first is based upon observations made by Maly[95]
many years ago, which tend to show that certain mineral salts present
in the blood are capable of reacting upon each other with formation
of hydrochloric acid. Thus, while the blood is an alkaline fluid,
it really owes its alkalinity to the presence of two acid salts,
viz., sodium bicarbonate (HNaCO_{3}) and disodium hydrogen phosphate
(HNa_{2}PO_{4}). This latter compound, acted upon by the carbonic
acid of the blood, is transformed into a dihydrogen sodium phosphate
with simultaneous formation of acid sodium carbonate, as shown in the
following equation:

    Na_{2}HPO_{4} + CO_{2} + H_{2}O = NaH_{2}PO_{4} + HNaCO_{3}.

[95] Untersuchungen über die Quelle der Magensaftsäure. Annalen d.
Chem. u. Pharm., Band 173, p. 227.

This acid sodium phosphate dissolved in a fluid containing sodium
chloride, gives rise to free hydrochloric acid by a very simple
reaction:

    NaH_{2}PO_{4} + NaCl = Na_{2}HPO_{4} + HCl.

It is also to be noted that the disodium hydrogen phosphate, may,
likewise, give rise to hydrochloric acid through its action on calcium
chloride, as indicated by the following equation:

    2Na_{2}HPO_{4} + 3CaCl_{2} = Ca_{3}(PO_{4})_{2} + 4NaCl + 2HCl.

It is thus evident that hydrochloric acid may originate in the
inter-reaction of these several salts which are known to be present
in the blood; but obviously, the above reactions cannot take place
in the blood itself, and we must look to the selective power of the
epithelial cells of the gastric glands, as suggested by Gamgee,[96] for
the withdrawal of the needed salts from the blood. Once present in the
acid-forming cells, and perhaps aided by the inherent qualities of the
protoplasm, the necessary chemical reactions may be assumed to take
place, after which the newly formed acid may pass from the gland-cells
into the secretion of the gland.

[96] Physiological Chemistry of the Animal Body, vol. 2, p. 113.

A later theory regarding the formation of the acid of the gastric juice
emanates from Liebermann.[97] This investigator claims the existence in
the mucous membrane of the stomach of an acid-reacting, nuclein-like
body, which is apparently a combination of the phosphorized substance
lecithin with a proteid. To this compound body Liebermann gives
the name of lecithalbumin. It is apparently located in the nuclei
of the gastric cells, is strongly acid in reaction, and, according
to Liebermann, is an important agent in the production of the free
hydrochloric acid of the gastric juice, although its action is somewhat
indirect. According to this theory, the free acid is formed in the
mucous membrane of the stomach from sodium chloride, through the
dissociating action of the carbonic acid coming from normal oxidation.
The thus-formed acid then diffuses in both directions, viz., through
the lumen of the gland into the stomach-cavity, and in part in the
opposite direction into the veins and lymphatics. It is the assumed
function of the lecithalbumin to react with the alkaline sodium
carbonate, produced simultaneously with the hydrochloric acid. This
naturally gives rise to the liberation of carbonic acid and to the
formation of a non-diffusible sodium-lecithalbumin compound, which
is retained for the time being in the body of the cell. When the
circulation of the blood, accelerated by the digestive process, returns
to its ordinary pace, this latter compound is slowly decomposed by
the carbonic acid with formation of the readily diffusible sodium
carbonate, which passes into the blood-current. The rate of this
latter reaction is impeded, or, perhaps regulated, by the swelling up
of the lecithalbumin-containing cells, thus rendering the imbibition
of the carbonic acid a slow process. The rate of production of the
hydrochloric acid by this hypothetical process depends primarily upon
the blood supply, and the oxidative changes by which carbonic acid is
formed.

[97] Studien über chemische Processe in der Magenschleimhaut. Pflüger’s
Archiv f. Physiol., Band 50, p. 25. Neue Untersuchungen über das
Lecithalbumin. Liebermann, Ibid., Band 54, p. 573.

There is much that might be said for and against this theory,[98] but
we cannot stop to discuss it here. Like the previous theory, it implies
the production of hydrochloric acid from a chloride or chlorides,
through chemical processes taking place in the stomach-mucosa, and
presumably in the large border-cells of the peptic glands. This
hydrochloric acid, as you know, in the act of secretion, reacts upon
the pepsinogen with which it may come in contact, transforming it into
pepsin. It also has the power of combining with all forms of proteid
matter, not excepting the products of proteolytic action, to form acid
compounds in which the so-combined acid, although equal quantitatively
to the original amount of free acid, is less active in many ways.
Thus, it does not possess in the same degree a destructive action on
the amylolytic ferments;[99] it does not play the same part in aiding
the proteolytic action of pepsin, and its antiseptic power is far from
equal to that of a like amount of free acid.[100]

[98] See discussion by Plósz and Liebermann in Jahresbericht für
Thierchemie, Band 22, p. 260.

[99] Chittenden and H. E. Smith: Studies in Physiol. Chem., Yale
Univer., vol. i., p. 18.

[100] Compare F. O. Cohn: Ueber die Einwirkung des künstlichen
Magensaftes auf Essigsäure-und Milchsäuregährung. Zeitschr. f. physiol.
Chem., Band 14, p. 74.

With relatively large amounts of proteid, we may have half or even
quarter saturated proteid molecules, in which the weakness of the
combined acid is far more pronounced than in the case of the fully
saturated molecule. Such a condition of things must obviously exist
in the early stages of gastric digestion. With an excess of proteid
matter in the stomach, some time must elapse before the secretion
of hydrochloric acid will be sufficient to furnish acid for all of
the proteid matter present, yet pepsin-proteolysis does not wait the
appearance of free acid. Indeed, the proteid matter may not have
combined with more than half its complement of hydrochloric acid before
digestive proteolysis is well under way. I have made many analyses of
the stomach-contents after test meals, and under other conditions,
where no free acid could be detected by the tropaeolin test, or better,
by Günzburg’s reagent (phloroglucin-vanillin), although phenolphthalein
as well as litmus showed strong acid reaction, and yet not only could
acid-albumin be detected in the filtered fluid, but likewise proteoses
and peptones. In other words, pepsin-proteolysis can proceed in the
absence of free hydrochloric acid, although not at the same pace.
Hence, proteoses and even peptones may make their appearance in the
stomach-contents at a very early period of digestion, _i. e._, the
final products of proteolysis may be found in a mixture containing even
a large proportion of wholly unaltered proteid, and obviously at an
early stage in the process. Expressed in other language, a portion of
the first formed acid-albumin or syntonin may be carried forward by the
digestive process to the secondary proteose and peptone stage, before
the larger portion of the ingested proteid food has even combined with
sufficient acid to insure the complete formation of acid-albumin.
This introduces another factor, to be referred to later on, viz.,
the relative combining power of different forms of proteid matter,
especially the proteoses and peptones, as contrasted with native
proteids.

In proof of the statement that pepsin-proteolysis can proceed in the
absence of free hydrochloric acid, provided combined acid be present,
allow me to cite one or two experiments bearing on this point. A
perfectly neutral solution of egg-albumen, containing 0.8169 gramme
of ash-free albumin per 10 c.c. of fluid, was employed as the proteid
material. In order to completely saturate the proteid contained in
20 c.c. of this neutral albumen solution, 50 c.c. of 0.2 per cent. HCl
were required. Two mixtures were then prepared as follows:

_A._ Twenty c.c. of the neutral albumen solution + 50 c.c. 0.2 per
cent. HCl + 30 c.c. of a weak aqueous solution of pepsin, perfectly
neutral to litmus. This mixture gave only the faintest tinge of a
reaction for free acid when tested by Günzburg’s reagent.

_B._ Twenty c.c. of the neutral albumen solution + 25 c.c. 0.2 per
cent. HCl + 30 c.c. of the neutral pepsin solution. In this mixture,
the proteid matter was obviously only half saturated with acid.

The two solutions were placed in a bath at 40° C., where they were
allowed to remain for forty-four hours, a little thymol being added to
guard against any possible putrefactive changes. At the end of this
time the amount of undigested albumin was accurately determined. The
20 c.c. of original albumen solution contained 1.6338 grammes of dry
coagulable albumin. At the end of the forty-four hours, _A_ contained
only 0.5430 gramme of unaltered albumin, or acid-albumin, while _B_
contained 1.2225 grammes. That is to say, in the mixture _A_, where the
acid existed wholly in the form of combined acid, but with the albumin
completely saturated, 1.0908 grammes of the proteid were converted
into soluble albumoses and peptones. In _B_, on the other hand, where
the albumin was only half saturated with acid, 0.4113 gramme of the
proteid was converted into soluble products. This difference in action
is made more striking by the statement that where the proteid was only
half saturated with acid, 25.1 per cent. of the albumin was digested;
while with a complete saturation of the proteid, 66.7 per cent. of the
albumin was digested.

To give emphasis to this matter, a second experiment may be quoted as
follows: The proteid used was the same neutral solution of egg-albumen
containing 0.8169 gramme of albumin per 10 c.c. Two mixtures were
prepared as follows:

_A._ Ten c.c. of the neutral albumen solution + 21.7 c.c. 0.2 per cent.
HCl, the amount needed to completely saturate the proteid, + 40 c.c. of
a weak solution of pepsin, perfectly neutral.

_B._ Ten c.c. of the albumen solution + 10.9 c.c. 0.2 per cent. HCl +
40 c.c. of the pepsin solution, making a mixture half saturated with
acid.

These two solutions were warmed at 40° C. for seventeen hours. The
extent of digestive action was then determined, when it was found that
in _A_ only 0.1638 gramme of the proteid was undigested, while in _B_,
0.6088 gramme remained unaltered. In other words, where the proteid was
completely saturated with acid, but with an utter lack of free acid,
79.9 per cent. of the albumin was converted into albumoses and peptone,
while in the mixture half saturated with acid only 25.4 per cent. was
digested.

These two experiments thus give striking proof that free acid is not
absolutely essential for pepsin-proteolysis. Digestion is, to be sure,
more rapid and complete when free hydrochloric acid is present, but
proteolysis is still possible, and even vigorous, when there is a
marked deficiency of free acid. Further, as we have seen, proteolysis
may proceed to a certain extent even though the amount of acid
available is not sufficient to combine with more than half the proteid
matter present.

These facts at once raise the question whether the products of
proteolysis may not have a stronger affinity for acid than the native
proteids; an affinity so strong that they may be able to withdraw acid
from the acid-albumin first formed. One of our conceptions regarding
pepsin-proteolysis is that acid is necessary for every step in the
proteolytic process. A primary albumose, for example, cannot be further
changed by pepsin, unless there is acid present for it to combine
with. This being true, it is clear, in view of the fact that even
peptones may appear in a digestive mixture containing an amount of
acid insufficient to combine even with the albumin present, that the
products of proteolysis must withdraw acid from the acid-albumin first
formed. In regard to the first point, my own experiments certainly
tend to show that the products of gastric digestion do combine with
larger amounts of hydrochloric acid than undigested proteids; and
further, that of the several products of proteolysis, the secondary
proteoses combine with a larger percentage of acid than the primary
proteoses, while true peptones combine with still larger amounts. In
other words, the simpler and more soluble the proteid, the larger the
amount of acid it is capable of combining with; a statement which
accords with results obtained by other workers[101] in this direction.
Further, another factor of considerable importance in connection
with the natural digestive process is that a dissolved proteid, such
as protoalbumose for example, will combine more readily with free
acid than an insoluble proteid; from which Gillespie[102] is led to
infer that in pepsin-proteolysis where there is no free acid present,
only acid-albumin, proteoses may be formed to a limited extent at
the expense of some of the acid of the acid-albumin, a portion of
the latter being perhaps reconverted into albumin. The ability of
the proteoses, however, to withdraw acid from its combination with a
native proteid is perhaps best indicated by Kossler’s[103] experiments,
which show that a solution of acid-albumin containing only enough
hydrochloric acid to hold the albumin dissolved, on being warmed at
40° C. for some hours with addition of a neutral solution of pepsin,
may undergo partial conversion into albumose or peptone.

[101] See especially Gillespie: Gastric Digestion of Proteids. Journal
of Anat. and Physiol., vol. 27, p. 207.

[102] Loc. cit.

[103] Beiträge zur Methodik der quantitativen Salzsäurebestimmung im
Mageninhalt. Zeitschr. f. physiol. Chem., Band 17, p. 93.

In spite of these facts, there is some evidence that while proteoses
and peptones have the power of combining with more acid than a like
weight of native proteid, the latter, leaving out all action of the
pepsin, has a stronger affinity for the acid; in fact, the firmness
or strength of the union appears to diminish as the products become
simpler.[104] Hence, a peptone separated from a digestive mixture, will
part with its combined acid somewhat more readily than acid-albumin
for example, although on this point there is not complete unanimity
of opinion.[105] In digestive proteolysis, however, where the pepsin
is accompanied by a minimal amount of hydrochloric acid, insufficient
perhaps to even half saturate the proteid present, the formation of
proteoses and peptones must be accompanied by a withdrawal of acid from
its combination with the native proteid.

[104] Compare Blum: Ueber die Salzsäurebindung bei künstlicher
Verdauung. Zeitschr. f. klin. Medicin, Band 21, p. 558.

[105] See Sansoni: Beitrag zur kenntniss des Verhaltens der Salzsäure
zu den Eiweisskörpern in Bezug auf die Chemische Untersuchung des
Magensaftes. Berliner klin. Wochenschrift, 1893, Nos. 42 and 43.

In illustration of some of these points, and especially of the
statement that the products of gastric digestion have the power of
combining with more hydrochloric acid than the original proteid, allow
me to cite the following experiment: 10 c.c. of a neutral solution of
egg-albumen containing about 0.82 gramme of pure dry albumin, free
from mineral salts, required 23.8 c.c. of 0.2 per cent. hydrochloric
acid to completely saturate the proteid matter. A mixture was then
prepared as follows: 10 c.c. of the albumen + 24 c.c. 0.2 per cent. HCl
+ 30 c.c. of a neutral pepsin solution, the mixture showing a faint
trace of free acid when tested by Günzburg’s reagent. This solution
was placed in a thermostat at 38° C., and from time to time a drop of
the fluid was removed and tested for free acid. If no reaction could
be obtained, 0.2 per cent. hydrochloric acid was added to the mixture,
until Günzburg’s reagent showed free acid to be again present. The
following table shows the rate of disappearance of free acid, and the
amounts of 0.2 per cent. HCl required to make good the deficiency.
The mixture was placed at 38° C. on February 6th, at 11.30 A.M., and,
as stated, contained a trace of free acid, 24 c.c. 0.2 per cent. HCl
having been added to accomplish this result.

             Time.           Acid added to show trace of free acid.
    February  6, 11.30 A.M.
        "         2.15 P.M.    4.5 c.c., 0.2 per cent. HCl.
        "         5.00 P.M.    1.0  "     "     "       "
    February  7,  8.45 A.M.    3.0  "     "     "       "
        "         2.00 P.M.    1.0  "     "     "       "
        "         5.00 P.M.    1.5  "     "     "       "
    February  8,  8.30 A.M.    1.0  "     "     "       "
        "         2.30 P.M.    0.0  "     "     "       "
    February  9,  8.30 A.M.    3.0  "     "     "       "
    February 10,  9.30 A.M.    2.0  "     "     "       "
                              ----
                              17.0

From these results several interesting conclusions may be drawn,
in conformity with the statements already made. Thus, as soon as
proteolysis commences, the products formed begin to show their greater
affinity for acid by withdrawing acid from its combination with the
native proteid, a supposition which is necessary to account for even
the starting of the proteolytic process. Further, it is evident that
proteoses and peptones combine with a far larger equivalent of acid
than the native albumin is capable of; 17 c.c. of 0.2 per cent. HCl
being required in the above experiment to satisfy the greater combining
power of the newly formed products. This doubtless depends upon the
cleavage of the large proteid molecule into a number of smaller or
simpler molecules, each of the latter, perhaps, combining with a
like number of HCl molecules. This view of the relationship of the
individual proteoses and peptones is one more or less generally held,
and is supported by many facts.[106] However this may be, it is evident
that the products of pepsin-proteolysis combine with a larger amount of
hydrochloric acid than the mother-proteid, and that the transformation
of the latter, at least under the conditions of this experiment,
is a slow and gradual process. In the living stomach, on the other
hand, where the secretion of acid is progressing with ever-increasing
rapidity, it is easy to see that the process of proteolysis would
naturally be much more rapid.

[106] See Gillespie: On the Gastric Digestion of Proteids. Journal of
Anatomy and Physiology, vol. 27, p. 209.

Just here we may recall the theory advanced by Richet[107] quite a
number of years ago that the acid of the gastric juice is a conjugate
acid, composed of leucin and hydrochloric acid, a theory which has
found little acceptance. Klemperer,[108] however, assumed that
solutions of leucin hydrochloride with pepsin would not digest albumin,
but Salkowski and Kumagawa[109] have shown by experiments that leucin
and other amido-acids, as glycocoll, may be dissolved in hydrochloric
acid in such proportion that the solution is practically composed of
leucin hydrochloride, without interfering with the digestive action
of pepsin-acid on blood-fibrin; the solution being physiologically
active, although Günzburg’s reagent shows an entirely negative result
for free acid. If the matter is studied quantitatively, however, it
will be found that the amido-acids combining in this manner with the
hydrochloric acid of the gastric juice do give rise to some disturbance
of proteolytic action;[110] _i. e._, digestion may be less rapid,
especially on egg-albumin, a conclusion which Salkowski[111] has lately
confirmed. Still, under such circumstances, digestion does go on and
at a fairly rapid rate; hence, if there is a combination between the
acid and these organic bodies, as is indicated by Günzburg’s reagent,
the acid is still active physiologically, even more so than in the
compound formed by the interaction of proteid and acid. In other
words, many of these neutral organic bodies that may originate in
the stomach through fermentative processes, or otherwise, and which
tend to combine with the acid of the gastric juice, do not, as a
rule, impede pepsin-proteolysis to the same extent that an excess of
proteid matter may. In fact, in artificial digestions long continued,
pepsin-acid solutions containing considerable leucin, for example, may
accomplish as much in the way of digesting proteid matter as the same
amount of pepsin-acid without leucin; but the inhibitory action of the
amido-acid is there, and may be shown during the first few hours of the
experiment, when less proteoses and peptones are formed than in the
control experiment without leucin.

[107] Le suc gastrique chez l’homme et les animaux, ses propriétés
chimiques et biologiques. Paris, 1878.

[108] Zeitschr. f. klin. Medicin, Band 14, Heft 1 and 2.

[109] Ueber den Begriff der freien und gebundenen Salzsäure im
Magensaft. Virchow’s Archiv, Band 122, p. 235.

[110] Rosenheim: Centralbl. f. klin. Medicin, 1891, No. 39. F. A.
Hofman, ibid., No. 42.

[111] Ueber die Bindung der Salzsäure durch Amidosäuren. Virchow’s
Archiv, Band 127, p. 501.

It is foreign to our subject to discuss here methods for the
detection of so-called free and combined hydrochloric acid in the
stomach-contents, or the special significance of such findings in
health and disease. I cannot refrain, however, in connection with what
has been said above concerning the proteolytic action of pepsin in the
presence of combined acid, from saying a word concerning the usual
deductions drawn from the absence of free acid in the stomach-contents.
As Langermann[112] has recently expressed it, we have methods for
discriminating between free and combined acid; we can, moreover,
determine the amount of free acid, but is it not equally important to
be able to say something definite concerning the amount of combined
acid in the stomach-contents? Even in the absence of free hydrochloric
acid there may be a sufficient amount of HCl secreted to answer all the
purposes of digestion, and yet at no time may there be any free acid
present to be detected by the various color-tests ordinarily made use
of. I am aware that in ordinary examinations of the stomach-contents
after a test meal the results are essentially comparative, and possibly
all that are necessary for clinical purposes. What I wish to emphasize,
however, is that in order to pass conclusively upon tsufficiency or
insufficiency of the gastric secretion, it is wise to know not only the
total acidity of the stomach contents and whether there is free acid
or not, but to know more about the amount of combined acid present.
Thus, there is a natural tendency to divide the fluids withdrawn from
the stomach into three groups, viz., those which contain free acid in
moderate amount, those which contain free acid in excess, and those
in which free acid is entirely absent; but in the latter group, there
may be very marked differences in the amount of acid combined with
the proteid and other material present. It appears to me that one of
the questions to be answered is whether there is sufficient combined
HCl present to meet all the requirements for digestion. If there
is, that gastric juice may be just as normal as the one containing
free mineral acid, and yet, according to our present tendencies,
we should be inclined to call the juice containing no free acid
abnormal, although there may be sufficient combined acid present to
meet all the requirements for digestion. Hence, in examination of the
stomach-contents, it is well to consider the use of those methods
which tend to throw light upon the amount of combined acid present,
for in my opinion it is only by a determination of the total amount of
combined acid that we can arrive at a true estimate of the extent of
the HCl deficiency. Obviously, in simple clinical examinations of the
stomach-contents after a test meal, where proteid matter is not present
in large amount, free acid may reasonably be expected to appear after
a definite period; but in any event, it is well to remember that free
hydrochloric acid is not absolutely indispensable for fairly vigorous
proteolytic action, and that in the presence of moderate amounts of
proteid matter a large quantity of acid is required to even saturate
the albuminous material.

[112] Virchow’s Archiv, Band 128, p. 408.

Consider for a moment the amount of acid a given weight of proteid
will combine with, before a reaction for free acid can be obtained.
Thus, Blum[113] has stated that 100 grammes of dry fibrin will require
9.1 litres of 0.1 per cent. hydrochloric acid to completely saturate
it. Hence, with a daily consumption of 100 grammes of proteid, there
would be needed for gastric digestion 4.5 litres of 0.2 per cent.
hydrochloric acid daily, and even this would not suffice to give any
free acid, assuming that none of the acid is used over again. The
results I have already given for egg-albumin tend to show that 1
gramme of pure albumin, free from inorganic salts, when dissolved in
a moderate amount of water will combine with about 30 c.c. of 0.2 per
cent. hydrochloric acid. Consequently, on this basis, 100 grammes of
dry egg-albumin will combine with 3 litres of 0.2 per cent. HCl, and
not until this amount of acid has been added to such a mixture will
reaction for free acid be obtained with Günzburg’s reagent. Hence
we can easily see, in view of these figures, that the production of
hydrochloric acid by the gastric glands may at times be very extensive,
without the stomach-contents necessarily containing free acid.

[113] Zeitschr. f. klin. Medicin, Band 21, p. 558.

While I am by no means willing to agree with Bunge[114] that the
chief importance of the acid of the gastric juice is its action as
an antiseptic, I am decidedly of the opinion that the lack of free
hydrochloric acid in the stomach-contents is more liable to cause
disturbance through the consequent unchecked development of bacteria
than through lack of proteolytic action, assuming, of course, the
presence of a reasonable amount of combined HCl. The hydrochloric
acid of the gastric juice unquestionably plays a very important part
in checking the growth and development of many pathogenic bacteria,
as well as of less poisonous organisms, which are taken into the
mouth with the food. On all, or at least on nearly all of these
organisms, hydrochloric acid exerts a far greater destructive action
when free than when combined with proteid matter. As Cohn[115] has
plainly shown, both hydrochloric acid and pepsin-hydrochloric acid
quickly hinder acetic- and lactic-acid fermentation, but when the
acid is combined with peptone, for example, it is no longer able to
exercise the same inhibitory influence. It is also important to note
that the lactic-acid ferment is not so sensitive to hydrochloric
acid as the acetic-acid ferment. Consequently, when lactic-acid
fermentation is once developed a comparatively large amount of HCl is
required to arrest it. Hence, as we all know, a diminished secretion
of hydrochloric acid renders possible acid fermentation of the
stomach-contents, as well as putrefactive changes which would not occur
in the presence of free HCl, and which are very incompletely checked
when the acid is over-saturated with proteid matter.

[114] Physiologische und Pathologische Chemie, p. 153.

[115] Ueber die Einwirkung des künstlichen Magensaftes auf
Essigsäure-und Milchsäure gährung. Zeitschr. f. physiol. Chemie, Band
14, p. 75. See also Hirschfeld: Pflüger’s Archiv f. Physiol., Band 47,
p. 510.

Pepsin-proteolysis, however, may proceed, to some extent, at least,
even though a small amount only of combined acid is present. The
combined acid, however, must be hydrochloric acid, if proteolysis is to
be at all marked. To be sure, pepsin will act in the presence of lactic
acid, as well as in the presence of other organic acids, and inorganic
acids, likewise, but such action at the best is considerably weaker
than the action of pepsin-hydrochloric acid.[116]

[116] Chittenden and Allen: Influence of Various Inorganic and
Alkaloidal Salts on the Proteolytic Action of Pepsin-Hydrochloric Acid.
Studies in Physiol. Chem., Yale University, vol. 1, pp. 91, 94.

The ferment pepsin can exert its _maximum_ action only in the
presence of free hydrochloric acid. There must be sufficient HCl to
combine with all of the proteid matter present, and the products of
proteolysis as fast as they are formed, if digestion is to be rapid
and attended with the formation of a large proportion of the final
products of proteolysis. It is under such conditions that our study
of pepsin-proteolysis is usually conducted. Further, it is to be
remembered that our knowledge of the products of such proteolytic
action depends almost entirely upon data accumulated by artificial
digestive experiments. In no other way can we be absolutely certain
of the conditions under which the proteolysis is accomplished, for it
is a significant fact, perhaps plainly evident from what has already
been said in the preceding lecture, that the character of the products
resulting from ordinary proteolysis is dependent in great part upon
the attendant circumstances. Thus, with a relatively small amount of
acid, and perhaps also of pepsin, the initial products of proteolysis
are especially prominent, while with an abundance of both pepsin and
free acid, coupled with long-continued action, the final products
predominate. Between these two extremes there are many possible
variations, as was, I think, made clear in the previous lecture. At
the same time, it is to be noticed that these differences are mainly
differences in the _proportion_ of the several products, rather than in
the nature of the resultant bodies.

In a general way, the products of pepsin-proteolysis may be divided
into three main groups, viz., bodies precipitated by neutralization and
represented mainly by the so-called syntonin or acid-albumin; bodies
precipitated by saturation of the neutralized fluid with ammonium
sulphate and represented by proteoses; bodies non-precipitable by
saturation with ammonium sulphate and represented by amphopeptones. The
relationship of the individual products may be clearly seen from the
following scheme, arranged after the plan suggested by Neumeister.

                NATIVE PROTEID.
                      |
                      |
                  Syntonin.
                   ╱    ╲
                 ╱        ╲
               ╱            ╲
       Protoproteose.   Heteroproteose.
             |           (dysproteose).
             |                 |
      Deuteroproteose.  Deuteroproteose.
             |                 |
          Peptone.          Peptone.

It is, of course, to be understood that this is not intended to
represent anything more than the order of formation of the several
bodies, no attention being paid here to the hemi- or anti-character
of the several products, or classes of products. Thus, proto and
heteroproteose are primary bodies formed directly from the initial
product syntonin by the further action of the ferment. In the same
sense, deuteroproteose is a secondary proteose, being formed by the
further hydration of the primary body. Lastly, peptones, the final
products of pepsin-proteolysis, are the result of the hydration and
possible cleavage of deuteroproteoses. Further, in almost every gastric
digestion there is also formed a small amount of antialbumid, a product
insoluble in dilute hydrochloric acid and which consequently appears
as an insoluble residue. This body is very resistant to the action of
pepsin-acid when once formed, but may be slowly converted, in part at
least, into a soluble antialbumose and thence into antipeptone.

All of these bodies can be readily identified in any digestive
mixture containing them by a few simple reactions. Thus, after having
removed any acid-albumin or syntonin present by neutralization, the
concentrated fluid can be tested at once. If primary proteoses are
present, the neutral fluid will yield a more or less heavy precipitate
on addition of crystals of rock-salt, precipitation being complete
only when the fluid is saturated with the salt. Further, if the
proteoses are present in not too small quantity, nitric acid added drop
by drop to the neutralized fluid will produce a white precipitate,
readily soluble on application of heat but reappearing as the solution
cools. If primary proteoses are wholly wanting, then no precipitate
will be obtained by acid unless the fluid is saturated with salt, in
which case a portion of the deuteroproteose will be precipitated.
The two primary proteoses differ from each other especially in
solubility; protoproteose being readily soluble in water alone, while
heteroproteose is soluble only in salt solutions, dilute acids, and
alkalies. Hence, when these two bodies are precipitated together by
saturation with salt, they may be readily separated by dissolving them
in a little dilute salt solution, and dialyzing the fluid in running
water until the salt is entirely removed; heteroproteose will then be
precipitated, while the proto-body remains in solution.

By long contact with water, and even with concentrated salt solutions,
heteroproteose tends to undergo change into a semi-coagulated form,
named dysproteose, insoluble in dilute sodium-chloride solutions.
This body can be reconverted into heteroproteose, in part at least,
by solution in dilute acid, or alkali, and reprecipitation by
neutralization.

As a class, the proteoses are characterized by far readier
solubility in water than native proteids, by a far greater degree of
diffusibility, by non-coagulability by heat and by alcohol, although
precipitable by the latter agent. Further, nearly all proteose
precipitates are exceedingly sensitive toward heat, tending to dissolve
as the fluid is warmed and reappearing as the solution cools. In
fact, this peculiarity often serves as a means of identification.
Potassium ferrocyanide and acetic acid, picric acid in excess, and
likewise cupric sulphate, all precipitate the primary proteoses, while
deuteroproteose is only slightly affected by these reagents, or indeed
not at all.

In order to separate the secondary proteose from the primary bodies
in the absence of peptones, the fluid is neutralized as nearly as
possible, and then, after suitable concentration, is saturated with
sodium chloride for the partial precipitation of the primary proteoses.
To the clear filtrate, acetic acid[117] is added drop by drop as long
as a precipitate results, the latter being composed of a mixture of
protoproteose and deuteroproteose. That is to say, protoproteoses
are not completely precipitated from neutral solutions by saturation
with salt alone; a little acid is required to complete it, but this
tends to bring down a certain amount of deuteroproteose. From this
filtrate, however, the deutero-body can be separated in a pure form by
dialyzing away the salt and acid, and then concentrating the fluid and
precipitating with alcohol. When the proteoses are mixed with peptones,
the former must first be separated collectively by saturation of the
fluid with ammonium sulphate.

[117] Saturated with sodium chloride.

Peptones are especially characterized by non-precipitation with the
ordinary precipitants for proteid bodies, and especially by the
fact that they are wholly indifferent to saturation with ammonium
sulphate either in neutral, acid, or alkaline fluids. This reaction,
which constitutes the main, and perhaps the only absolute method
of separating peptones from proteoses must be carried out with
great thoroughness in order to insure a complete precipitation of
deuteroproteose. The latter stands midway between primary proteoses and
peptones in many respects, and seems to share with peptones something
of a tendency to resist precipitation by the ammonium salt. Indeed, as
Kühne[118] has recently pointed out, the last traces of deuteroproteose
can be precipitated from the fluid only by long continued boiling of
the ammonium sulphate-saturated fluid, and even then it is seldom
complete unless the reaction of the fluid is alternately made neutral,
acid, and alkaline, and the heating continued for some time after each
change in reaction. Under such circumstances, the last portions of
deuteroproteose separate from the salt-saturated fluid and float on the
surface in the form of an oily or gummy mass, while the true peptone
remains in the fluid absolutely non-precipitable by the salt.

[118] Erfahrungen über Albumosen und Peptone. Zeitschr. f. Biol., Band
29, p. 2.

In this filtrate, peptone can be detected by adding to a small portion
of the fluid a very large excess of a strong solution of potassium
hydroxide, followed by the addition of a few drops of a very dilute
solution of cupric sulphate. If peptone is present a bright red color
will appear, the intensity of which, with the proper amount of cupric
sulphate, will be proportional to the amount of peptone present. If it
is desired to separate the peptone from the ammonium-sulphate-saturated
fluid, there are several methods available, of which the following is
perhaps the most satisfactory: The fluid is concentrated somewhat,
and set aside in a cool place for crystallization of a portion of the
ammonium salt. The fluid is then mixed with about one-fifth its volume
of alcohol, and allowed to stand for some time, when it separates
into two layers--an upper one, rich in alcohol, and a lower one, rich
in salts. The latter is again treated with alcohol, by which another
separation of the same order is accomplished. Finally, the lighter
alcoholic layers containing the peptone are united, and exposed to a
low temperature until considerable of the contained salt crystallizes
out. The fluid is then concentrated, and after addition of a little
water is boiled with barium carbonate until the fluid is entirely
free from ammonium sulphate. Any excess of baryta in the filtrate is
removed by cautious addition of dilute sulphuric acid, after which the
concentrated fluid, reduced almost to a sirupy mass, is poured into
absolute alcohol for precipitation of the peptone.

So separated, the peptone formed in gastric digestion is exceedingly
gummy, but can be transformed into a yellowish powder, very
hygroscopic, of more or less bitter taste, and, when thoroughly dry,
dissolving in water with a hissing sound and with considerable
development of heat, like phosphoric anhydride.[119]

[119] Kühne and Chittenden: Peptones. Studies in Physiol. Chem., Yale
University, vol. 2, p. 14.

I have introduced these dry chemical facts, none of which are
especially new, because I deem them of considerable importance and
because they are not very generally known. In fact, there seems to be
a tendency on the part of some who are more or less familiar with the
advances made in our knowledge of the products of pepsin-proteolysis
to question the existence of these different bodies, or to show at
least a spirit of indifference toward these recent facts which have
been gradually accumulated, and I may say accumulated at the expense
of considerable labor. The time is past for calling the products of
gastric digestion peptones; it is time for a full recognition of the
fact that pepsin-proteolysis is synonymous with the production of a row
of bodies, chemically and physiologically distinct from each other,
each endowed with individuality enough to admit of certain detection,
and all bearing a certain specific and harmonious relationship to their
neighbors, the other members of the series.

Further, it is not enough to admit the formation of a single
intermediate body, midway between syntonin and peptone. The so-called
propeptone of the past is simply a mixture of proteoses, of ever
changing composition, varying with each change in the proportion of the
component proteoses. Each of these proteoses can be detected, under
suitable conditions, in the products of every artificial digestion
as well as in the stomach-contents, and no better measure of the
proteolytic power of the natural stomach-secretion can be devised
than a study of the character of the individual bodies present in the
stomach-contents after a suitable test meal. The proper tests and
separations can be made with a small amount of the filtered fluid, and
much light thrown upon the digestive power of the secretion by even a
rough estimate of the proportion of primary and secondary proteoses
and peptones formed in a given time, after the ingestion of a certain
amount of proteid food.

In pepsin-proteolysis we have to deal, in my opinion, with a series of
progressive hydrolytic changes in which peptones are the final products
of the transformation. Commencing with the formation of acid-albumin
or syntonin, hydrolysis and cleavage proceed hand in hand, under the
guiding influence of the proteolytic enzyme, and each onward step in
the process is marked by the appearance of a new body corresponding to
the extent of the hydrolysis; each body, perhaps, being represented
by a row or series of isomers, all externally alike, but different
in their inner structure, according to the proportion of hemi- and
anti-groups contained in the molecule. As opposed to this theory, we
have the older views of Maly,[120] Herth,[121] Henninger[122] and
others, based upon observations which tend to show that peptones do
not differ in chemical composition from the proteids which yield them.
As a matter of fact, the products then analyzed were not peptones at
all; they were merely the _primary_ products of pepsin-proteolysis,
_i. e._, what we now term primary proteoses, and it is time we stopped
using such data to enforce the theory that peptones are polymers of the
proteids from which they are derived.

[120] Ueber die chemische Zusammensetzung und physiologische Bedeutung
der Peptone. Pflüger’s Archiv f. Physiol., Band 9, p. 585.

[121] Ueber die chemische Natur des Peptones und sein Verhältniss zum
Eiweiss. Zeitschr. f. physiol. chem., Band 1, p. 277.

[122] De la Nature et du rôle physiologique des peptones. Paris, 1878.

In 1886, the writer, in conjunction with Professor Kühne, commenced
a study of the various cleavage products[123] formed by the action
of pepsin-hydrochloric acid from the better characterized and
purer proteids, this being a continuation of our earlier work on
the proteoses and peptones formed from blood-fibrin, serum-albumin,
etc. This work I have continued in my laboratory up to the present
time, with many co-workers, and as a result we have to-day a series
of observations gradually accumulated during these last seven years,
some the results of work carried on this last year, which speak in
no uncertain way of the character of both the primary and secondary
products of pepsin-proteolysis. Furthermore, in attempting to settle
this question once for all, I have selected for study examples from
the various classes of both animal and vegetable proteids; and as
representatives of the latter have had carried out two lengthy series
of experiments on the crystallized proteids which occur so abundantly
in some seeds, on the assumption that these crystalline bodies would
furnish a certain guarantee of purity which might naturally be lacking
in the amorphous proteids of animal origin. Some of these results are
now placed together in the following tables, a study of which reveals
some very interesting facts:

[123] Globulin and Globuloses. Studies in Physiol. Chem. Yale
University, vol. ii., p. 1.


COMPOSITION OF PROTEOLYTIC PRODUCTS FORMED BY PEPSIN-HYDROCHLORIC ACID.


_Proteolysis of Blood-fibrin._

    =====================================================
      | Mother |  Proto-  |  Hetero- | Deutero- | Ampho-
      |Proteid.|fibrinose.|fibrinose.|fibrinose.|peptone.
      |        |  [124]   |  [124]   |  [124]   | [125]
    --+--------+----------+----------+----------+--------
    C | 52.68  |  51.50   |  50.74   |  50.47   | 48.75
    H |  6.83  |   6.80   |   6.72   |   6.81   |  7.21
    N | 16.91  |  17.13   |  17.14   |  17.20   | 16.26
    S |  1.10  |   0.94   |   1.16   |   0.87   |  0.77
    O | 22.48  |  23.63   |  24.24   |  24.65   | 27.01
    --+--------+----------+----------+----------+--------

[124] Kühne and Chittenden: Zeitschr. f. Biol., Band 20, p. 40.

[125] Kühne and Chittenden: Studies in Physiol. Chem., Yale Univer.,
vol. ii., p. 40.


_Proteolysis of Paraglobulin._[126]

    ============================================
      | Mother |  Proto-  | Hetero-  | Deutero-
      |Proteid.|globulose.|globulose.|globulose.
    --+--------+----------+----------+----------
    C | 52.71  |  51.57   |  52.10   |  51.52
    H |  7.01  |   6.98   |   6.98   |   6.95
    N | 15.85  |  16.09   |  16.08   |  15.94
    S |  1.11 }|          |          |
    O | 23.24 }|  25.36   |  24.84   |  25.59
    --+--------+----------+----------+----------

[126] Kühne and Chittenden: Studies in Physiol. Chem., Yale Univer.,
vol. ii, p. 12.


_Proteolysis of Coagulated Egg-albumin._

    ==================================================
      | Mother |  Proto- | Hetero- |Deutero- | Hemi-
      |Proteid.|albumose.|albumose.|albumose.|peptone.
      |        |  [127]  |  [127]  |  [127]  | [128]
    --+--------+---------+---------+---------+--------
    C | 52.33  |  51.44  |  52.06  |  51.19  | 49.38
    H |  6.98  |   7.10  |   6.95  |   6.94  |  6.81
    N | 15.84  |  16.18  |  15.55  |  15.77  | 15.07
    S |  1.81  |   2.00  |   1.63  |   2.02  |  1.10
    O | 23.04  |  23.28  |  23.81  |  24.08  | 27.64
    --+--------+---------+---------+---------+--------

[127] Chittenden and Bolton: _Ibid._, vol. ii, p. 153.

[128] Kühne and Chittenden: Zeitschr. f. Biol., Band 19, p. 201.


_Proteolysis of Casein from Milk._

    ===================================================
      | Mother | Proto- |Hetero- |α Deutero-|β Deutero-
      |Proteid.|caseose.|caseose.| caseose. | caseose.
      |        | [129]  | [130]  |  [129]   |  [129]
    --+--------+--------+--------+----------+----------
    C | 53.30  | 54.58  | 53.88  |  52.10   |  47.72
    H |  7.07  |  7.10  |  7.27  |   6.93   |   6.73
    N | 15.91  | 15.80  | 15.67  |  15.51   |  15.97
    S |  0.82 }|        |        |          |
    O | 22.03 }| 22.52  | 23.18  |  25.46   |  29.58
    --+--------+--------+--------+----------+----------

[129] Chittenden: Studies in Physiol. Chem., Yale Univer., vol. iii.,
p. 80.

[130] Chittenden and Painter: _Ibid._, vol. ii., p. 195.


_Proteolysis of Myosin from Muscle._[131]

    ====================================================
      |Mother Proteid.|Protomyosinose.|Deuteromyosinose.
    --+---------------+---------------+-----------------
    C |     52.82     |     52.43     |     50.97
    H |      7.11     |      7.17     |      7.42
    N |     16.77     |     16.92     |     17.00
    S |      1.27     |      1.32     |      1.22
    O |     21.90     |     22.16     |     23.39
    --+---------------+---------------+-----------------

[131] Kühne and Chittenden: _Ibid._, vol. iii, p. 147.


_Proteolysis of Elastin._[132]

    ==+===============================================
      |Mother Proteid.|Protoelastose.|Deuteroelastose.
    --+---------------+--------------+----------------
    C |    54.24      |    54.52     |     53.11
    H |     7.27      |     7.01     |      7.08
    N |    16.70      |    16.96     |     16.85
    S}|               |              |
    O}|    21.79      |    21.51     |     22.96
    --+---------------+--------------+----------------

[132] Chittenden and Hart: Studies in Physiol. Chem., Yale Univer.,
vol. iii, p. 37.


_Proteolysis of Gelatin._[133]

    ==+===============================================
      |Mother Proteid.|Protogelatose.|Deuterogelatose.
    --+---------------+--------------+----------------
    C |    49.38      |    49.98     |     49.23
    H |     6.81      |     6.78     |      6.84
    N |    17.97      |    17.86     |     17.40
    S |     0.71      |     0.52     |      0.51
    O |    25.13      |    24.86     |     26.02
    --+---------------+--------------+----------------

[133] Chittenden and Solley: Journal of Physiol., vol. xii, p. 33.


_Proteolysis of Phytovitellin_[134] _(Crystallized) from Squash Seed._

    ==+===============+===============+=================
      |Mother Proteid.|Protovitellose.|Deuterovitellose.
    --+---------------+---------------+-----------------
    C |    51.60      |     51.52     |     49.27
    H |     6.97      |      6.98     |      6.70
    N |    18.80      |     18.67     |     18.78
    S |     1.01}     |               |
    O |    21.62}     |     22.83     |     25.25
    --+---------------+---------------+-----------------

[134] Chittenden and Hartwell: _Ibid._, vol. xi, p. 441.


_Proteolysis of Phytovitellin_[135] _(Crystallized) from Hemp Seed._

    =============================================================
      |Mother Proteid.|Protovitellose.|Deuterovitellose.|Peptone.
    --+---------------+---------------+-----------------+--------
    C |     51.63     |    51.55      |     49.78       | 49.40
    H |      6.90     |     6.73      |      6.73       |  6.77
    N |     18.78     |    18.90      |     17.97       | 18.40
    S |      0.90     |     1.09      |      1.08       |  0.49
    O |     21.79     |    21.73      |     24.44       | 24.94
    --+---------------+---------------+-----------------+--------

[135] Chittenden and Mendel: _Ibid._, vol. xvii, p. 48.


_Proteolysis of Glutenin_[136] _from Wheat._

    ==+=========================================
      | Mother |  Proto-  | Hetero-  | Deutero-
      |Proteid.|glutenose.|glutenose.|glutenose.
    --+--------+----------+----------+----------
    C | 52.34  |  51.42   |  51.82   |  49.85
    H |  6.83  |   6.70   |   6.79   |   6.69
    N | 17.49  |  17.56   |  17.43   |  17.57
    S |  1.08  |   1.34   |   1.59   |   0.80
    O | 22.26  |  22.98   |  22.37   |  25.09
    --+--------+----------+----------+----------

[136] Formerly called gluten-casein, and the products gluten-caseoses.
Chittenden and E. E. Smith: Journal of Physiol., vol. xi, p. 420.


_Proteolysis of Zein._[137]

    ============================================
      |Mother Proteid.|Protozeose.|Deuterozeose.
    --+---------------+-----------+-------------
    C |     55.23     |   53.29   |    51.31
    H |      7.26     |    6.87   |     6.88
    N |     16.13     |   16.10   |    16.27
    S |      0.60     |    1.54   |     1.08
    O |     20.78     |   22.20   |    24.46
    --+---------------+-----------+-------------

[137] Chittenden and Williams: Not heretofore published.

In considering these results, it is to be noticed that there is a
general unanimity of agreement except in the case of the albuminoid
gelatin. In the proteolysis of this body, for some reason not
explainable, the digestive products show no marked deviation from the
composition of the mother-proteid, but in every other instance there
is to be traced a distinct tendency toward diminution in the content
of carbon, proportional to the extent of proteolysis. In the primary
bodies, proto and heteroproteoses, the percentage of carbon is only
slightly lowered; indeed, in some few cases, notably in elastin and
casein, the primary products show a slight increase in their content
of carbon, but in most instances there is a slight falling off in the
percentage of this element. In the deuteroproteoses, however, the loss
of carbon is very marked. The percentage loss, to be sure, varies with
the different proteids, doubtless dependent in part upon the nature
of the proteid itself, and also, I think, upon the strength of the
proteolytic agent employed and the duration of the proteolysis. It is
to be further noticed that peptones, whenever analyzed, show a still
further loss of carbon and also a marked loss of sulphur. In nitrogen
there is no constant difference.

On the assumption that these various products of proteolysis are
formed by a series of hydrolytic changes, accompanied by cleavage of
the molecule, we might at first glance look for a marked increase in
the content of hydrogen. But when we consider the size of the proteid
molecule, with the small proportion of hydrogen contained therein
and the large amount of carbon, it is plain that hydrolytic cleavage
might naturally leave its mark on the percentage of carbon, rather
than on the percentage of hydrogen of the resultant products. In view
of these facts, the above results show nothing inconsistent with the
theory that pepsin-proteolysis, as a rule, is accompanied by a series
of progressive hydrolytic cleavages in which the primary proteoses are
the result of a slight hydration, these bodies by continued proteolysis
being further hydrated with formation of secondary proteoses, which in
turn undergo final hydration and cleavage into true peptones. In accord
with this theory, true peptones always show a marked difference in
composition from that of the mother-proteid, the most striking feature
being the greatly diminished content of carbon, which may be taken as
a measure, in part at least, of the extent of the hydrolytic change.
And it is to be noticed that the crystallized phytovitellins are no
exception to the general rule; the secondary vitelloses and peptones
resulting from proteolysis bear essentially the same relationship to
the mother-proteids that the albumoses from egg-albumin do. Moreover,
the alcohol-soluble proteids, of which the zein of cornmeal is a good
example, show the same general tendency, and it is an interesting
fact that the proteoses, or more specifically the zeoses, formed from
this peculiar proteid, are readily soluble in water and show the
general proteose reactions. It may also be mentioned that these zeoses,
as well as the elastoses, are very resistant to further hydrolysis
by pepsin-acid, and yield only comparatively small amounts of true
peptones.

In connection with this question of the composition of proteoses
and peptones as formed by pepsin-proteolysis, it is interesting to
note a recent observation recorded by Schützenberger.[138] This
experimenter took 350 grammes of moist blood-fibrin, corresponding to
75.5 grammes of dry substance, and subjected it to proteolysis with
2.5 litres of a very strong pepsin-hydrochloric acid solution for five
days. The resultant fluid was then freed from acid by treatment with
silver oxide, after which the solution was evaporated to dryness on
a water-bath and the residue dried _in vacuo_. This residue, termed
by Schützenberger fibrin-peptone, was found on analysis to contain
49.18 per cent. of carbon, 7.09 per cent. of hydrogen, and 16.33 per
cent. of nitrogen, thus agreeing very closely with true fibrin-peptone
as analyzed by Kühne and myself. Further, Schützenberger showed that
the fibrin in undergoing this transformation had taken on 3.97 per
cent. of water. But to my mind, the most significant fact connected
with this experiment is the positive evidence it affords, not only of
hydration as a feature of peptonization by pepsin-acid, but that this
greatly diminished content of carbon, so characteristic of peptones,
and to a less extent of deuteroproteoses, is wholly independent of the
methods of separation and purification ordinarily made use of. Thus,
Schützenberger, in the above experiment, did not attempt any separation
of individual bodies. Proteolysis was carried out under conditions
favoring maximum conversion into peptone, and the resultant product,
or products, was analyzed directly without recourse to any methods
of precipitation or purification. To be sure, the substance analyzed
could not have been peptone entirely free from proteose, but in any
event it represented the terminal products of pepsin-proteolysis, and
like true amphopeptone contained 3.5 per cent. less carbon than the
original fibrin. Hence, we may conclude, without further argument, that
peptonization in gastric digestion is the result of distinct hydrolytic
action, in which the original proteid molecule is gradually broken
down, or split apart, into a number of simpler molecules, the proteoses
and peptones.

[138] Recherches sur la constitution chimique des peptones. Comptes
Rendus, vol. 115, p. 208.

Peptones, _i. e._, amphopeptones, are the final products of gastric
digestion; but to how great an extent is actual peptonization carried
on in pepsin-proteolysis? As we have seen, syntonin, primary proteoses,
secondary proteoses, and peptones are all products of pepsin-digestion,
and it might perhaps be assumed that ultimately all of a given proteid
undergoing pepsin-proteolysis would be converted into amphopeptone.
Examination, however, shows that such is not the case, at least in
artificial digestive experiments. Peptones are truly formed, and
many times in large amount, but never under any circumstances have
I been able to effect a complete transformation of any proteid into
true peptone by pepsin-proteolysis; there is always found a certain
amount of proteoses more or less resistant to the further action of
the ferment. Obviously, the nature and proportion of the individual
products formed in any digestive experiment are dependent greatly
upon the attendant conditions; but even with a large amount of active
ferment, an abundance of free hydrochloric acid, a proper temperature,
and a long-continued period of digestion, even five and six days,
there is never found a complete conversion into peptone. Indeed,
the largest yield of peptone I have ever obtained in an artificial
digestion is sixty per cent., while the average of a large number of
results under most favorable circumstances is somewhat less than fifty
per cent.[139]

[139] Chittenden and Hartwell: The Relative Formation of Proteoses and
Peptones in Gastric Digestion. Journal of Physiol., vol. xii, p. 12.

We understand that peptones are the products of the hydration and
cleavage of previously formed proteoses. The primary proteoses pass
into secondary proteoses and these into peptones, but for some reason
this transformation after a time becomes a slow and gradual process. At
first there is a marked and rapid progression; the proteid undergoing
proteolysis is rapidly dissolved, and both proteoses and peptones may
be detected in abundance. But if we continue to watch the changing
relations of primary and secondary proteoses and peptones, we find
that progression soon ceases to be rapid, and eventually travels
onward at a snail’s pace. Thus, in one experiment with coagulated
egg-albumin, there was found at the end of forty-eight hours’ digestion
with pepsin-hydrochloric acid, only thirty-seven per cent. of peptones
with fifty-eight per cent. of proteoses, and yet digestion had been
sufficiently vigorous to allow of a complete solution of the proteid
in two hours. At the end of seventy-two hours the amount of peptones
had increased to about forty-two per cent., the proteoses having
correspondingly diminished; but even at the end of seventeen days only
fifty-four per cent. of peptones were to be found, thus affording
striking evidence of the slow conversion of the first-formed products
into peptones.

Naturally, the individual proteoses show marked differences in their
rate of conversion into secondary or final products. Take as an
illustration some results[140] obtained with caseoses formed in the
digestion of the casein of milk. Thus, heterocaseose, a primary
product, yielded only fifteen per cent. of peptone after ninety-four
hours at 40° C. with a strong pepsin-acid solution. Protocaseose,
however, containing some deuterocaseose, under like conditions, yielded
thirty-two per cent. of peptone in one hundred and nineteen hours,
while pure deuterocaseose gave sixty-six per cent. of peptone in one
hundred and thirty-seven hours. Evidently, then, the first-formed
soluble products of gastric digestion, _i. e._, the primary proteoses,
are only slowly converted into peptone, since they must first pass
through the intermediate stage of deuteroproteose, which is plainly
not a rapid process. The deutero-body, on the other hand, once formed
is more rapidly converted into peptone, but even this is in no sense
a rapid process. Hence, in the artificial digestion of proteids with
pepsin-hydrochloric acid, solubility of the proteids may be quite
rapid, and even complete in a very short time, but the resultant
products will be mainly proteoses and not peptones. The latter are
truly formed and in considerable amount, but proteoses, either as
primary or secondary bodies, are invariably present and usually in
excess of the peptones.

[140] Chittenden and Hartwell, loc. cit., p. 22.

In this connection the question naturally arises how far we are
to trust these results in their bearing on the natural process of
digestion as it occurs in the living stomach. Obviously, the conditions
are quite different in the two cases. In artificial digestions, we
have especially the influence of an ever-increasing percentage of
soluble products on the activity of the ferment, a condition of things
generally considered as more or less inhibitory to enzyme action.
We have attempted to measure the real value of this influence by
experiments[141] conducted in parchment dialyzing tubes, in which
the conditions are made favorable for the removal of at least some
of the products of digestion as fast as they are formed. In these
experiments, the dialyzer tubes containing the proteid and pepsin-acid
were immersed in a large volume of 0.2 per cent. hydrochloric acid
(about three litres), which was gradually changed from time to time,
the whole mixture being kept at 40° C. during the entire period of
the experiment. The extent of peptonization was then ascertained
by analysis of both the contents of the dialyzer tubes and of the
surrounding acid, the results being compared with those obtained from
control experiments carried on in flasks. Without considering the
results in detail, it may be mentioned that the slow and incomplete
peptonization so characteristic of artificial gastric digestion is not
materially modified by this closer approach to the natural process.
The several digestions carried on in the dialyzer tubes were certainly
accompanied by a fairly rapid withdrawal of the diffusible products of
digestion, yet no noticeable increase in the amount of peptone formed
was observed. The results certainly favor the view that the conversion
of the primary products of gastric digestion into true peptone is a
slow and gradual process, even under the most favorable circumstances,
and that this lack of complete peptonization is not due to accumulation
of the products of digestion, but is rather an inherent quality of
pepsin-proteolysis under all circumstances.

[141] Chittenden and Amerman: A Comparison of Artificial and Natural
Gastric Digestion, together with a Study of the Diffusibility of
Proteoses and Peptones. Journal of Physiol., vol. xiv, p. 483.

In these dialyzer experiments it was observed that not only did
peptones diffuse, but also the proteoses. In fact, it was found that
six to eight per cent. of the proteoses formed passed through the
parchment walls of the dialyzer tubes into the surrounding acid in
the nine hours’ digestion. This led to a study of the diffusibility
of proteoses in general, from which we were led to conclude that
these bodies possess this power to a greater degree than had hitherto
been supposed. As might be expected, it was also found that the
attendant conditions modify materially the rate of diffusibility; the
two factors especially prominent being temperature and the volume of
the surrounding fluid. Thus, 1.9 grammes of protoalbumose dissolved
in 200 c.c. of water and suspended in 4.5 litres of water heated to
38° C., diffused through the parchment tube to the-extent of 5.09 per
cent., while at 10° C. diffusion amounted to only 2.57 per cent. Under
somewhat similar conditions, pure peptone diffused to the extent of
eleven per cent. in six hours at 38° C. Somewhat singular, however,
was the result obtained with deuteroalbumose; this proteose showing a
diffusibility considerably less than that of the proto-body. But as
Kühne[142] has independently obtained essentially the same results,
this apparent anomaly cannot depend upon any errors of work.

[142] Erfahrungen über Albumosen und Peptone. Zeitschr. f. Biol., Band
29, p. 20.

It is of course to be understood that diffusion experiments made with
dead parchment membranes cannot necessarily be expected to throw much
light upon the rate of absorption of these bodies through the living
membranes of the stomach and intestine, where, as Waymouth Reid[143]
has well said, we have to deal with an absorptive force dependent, no
doubt, upon protoplasmic activity, and comparable, in part at least,
to the excretive force of a gland-cell. Furthermore, in considering
absorption as it occurs in the living stomach, we must necessarily
give due weight to the selective power of the epithelial cells,
a power which may be far more potent even than we suppose. Hence,
without attempting at this point to draw any broad deductions from our
experiments we may simply lay stress upon the facts themselves, viz.,
that the primary products of pepsin-proteolysis are diffusible, and,
like true peptones, are capable of passing through animal and vegetable
membranes, although to a less extent. We may further emphasize the
fact that experiments of this character on diffusibility can, at the
most, only indicate general tendencies, since every variation in the
attendant conditions will exercise some influence upon the final result.

[143] Osmosis Experiments with Living and Dead Membranes. Journal of
Physiol., vol. xi, p. 312.

With reference to the bearing digestive experiments made in dialyzer
tubes have upon the natural process as carried on in the living
stomach, we must necessarily grant that the conditions approximate
only in the crudest way to those existent in the alimentary tract.
At the same time, if complete peptonization is characteristic of
pepsin-proteolysis in the stomach, and failure to obtain such results
in an artificial digestion is due to lack of withdrawal of the
diffusible products formed, then certainly the experiments carried on
in dialyzer tubes, with abundant opportunity for diffusion, and with a
large excess of free hydrochloric acid, should show some indications of
increased peptone-formation. But none were obtained.

It is more than probable that the rate of absorption of diffusible
products from the stomach has been overestimated. Lea,[144] for
example, assumes that, “normally the products of digestion, whether
proteid or carbohydrate, are never met with in either the stomach
or intestine in other than the smallest amounts, frequently to be
described as merely traces.” This certainly implies a far more rapid
absorption of proteoses and peptones from the stomach than results
seem to justify. Indeed, recent facts obtained by Brandl,[145] working
under Tappeiner’s direction, tend to show that absorption from the
stomach is, under some circumstances at least, comparatively slow.
Brandl’s experiments were conducted on large and vigorous dogs with
gastric fistulæ, the stomach being shut off from the intestine by the
simple introduction of a small rubber balloon into the pylorus, which
when dilated completely closed the orifice. By carefully conducted
experiments, it was shown that pure peptone, entirely free from
proteose, is absorbed from the empty stomach in proportion to the
concentration of the peptone solution. Thus, 7.5 grammes of peptone
dissolved in water in such proportion as to make a five per cent.
solution, and allowed to remain in the stomach for two hours, lost by
absorption only 0.28 gramme, equal to 2.68 per cent. of the peptone
introduced. Under similar conditions, a ten per cent. aqueous solution
of peptone lost only 4.5 per cent. by absorption. On the other hand,
when peptone was introduced in larger quantity, viz., in a twenty per
cent. solution, absorption amounted to thirteen per cent. in two hours.

[144] Journal of Physiol., vol. xi, p. 240.

[145] Ueber Resorption und Secretion im Magen, und deren Beeinflussung
durch Arzneimittel. Zeitschr. f. Biol., Band 29, p. 277.

It is thus evident that pure peptones, even when taken into the stomach
in fairly large amounts, and under conditions very favorable for rapid
absorption, pass into the circulating blood very slowly. Obviously,
however, one must not lose sight of the fact that when digestion is
under way and the volume of blood consequently increased, there may
be a corresponding rise in the rate of absorption. There is perhaps a
hint of this conclusion in the influence of alcohol on the absorption
of peptone as brought out by some of Brandl’s experiments. Thus, it
was found that when alcohol was added in considerable quantity to a
ten per cent. solution of peptone, the stomach-mucosa was greatly
reddened, while in two hours the absorption of peptone amounted to
11.8 per cent. But in any event, these results certainly do not favor
the view that the products of gastric digestion are absorbed as soon
as they are formed. It is, no doubt, quite different in the intestine,
but in the stomach, where pepsin-proteolysis occurs, we have, I think,
no grounds for assuming that either peptones or proteoses are rapidly
absorbed. Hence, it might perhaps be considered that the results of
pepsin-proteolysis in the living stomach are much the same as those
obtained in artificial digestion experiments.

Still, there are other differences between natural digestion and
artificial proteolysis than those connected with the possible
absorption of the more diffusible products of digestion. Thus, in the
living stomach there is an ever-increasing secretion of hydrochloric
acid, and perhaps also of pepsin, more or less proportional to the
extent of proteolysis. On this point Brandl’s experiments again give
us some light. Thus, it was found that the introduction of an aqueous
solution of peptone into the empty stomach led to the secretion of an
acid fluid containing on an average 0.24 per cent. HCl, while, under
similar conditions, the introduction of sugar or potassium iodide
was followed by the secretion of a fluid containing on an average
only 0.13 per cent. HCl. Further, the absolute amount of acid found
after the introduction of peptone was far greater than when sugar or
iodide was introduced, since peptone led to an increase of at least
fifty per cent. in the volume of fluid secreted. Hence, proteolysis in
the living stomach may give rise to such an increased production and
secretion of hydrochloric acid that formation of the terminal products
of gastric digestion may be greatly accelerated. That such in fact is
the case, I have no manner of doubt, but that it may result in the
complete conversion of the so-called primary and secondary proteoses
into peptone I very much question. In fact, such examinations as I have
made of the stomach-contents after a suitable test-meal have always
resulted in the finding of a relatively large amount of proteoses. To
be sure, true peptone may be detected and in fairly large amounts,
but whenever a quantitative determination of the relative proportion
of the two has been made, the proteoses have always been in excess. I
have already reported elsewhere the results of some experiments in this
direction made on a healthy young man, where the stomach-contents were
withdrawn at varying periods after the ingestion of weighed amounts
of coagulated egg-albumin. Thus, in one experiment[146] the stomach
was thoroughly rinsed with water, after which 138 grammes of finely
divided coagulated-albumin, equal to 16 grammes of dry albumin, were
ingested. Three-quarters of an hour thereafter, the stomach-contents
were withdrawn by lavage and analyzed. As a result, 1.41 grammes of
albumoses were separated and weighed, and 0.84 gramme of peptones,
the relative proportion being expressed by sixty-two per cent. of
albumoses and thirty-seven per cent. of peptones, calculated on the
2.25 grammes of soluble products recovered. This expresses the general
character of the results obtained in experiments of this nature, and in
my opinion adds emphasis to the statement already made, that complete
peptonization is not a feature of pepsin-proteolysis, either in the
artificial or in the natural process as it takes place in the living
stomach.

[146] Journal of Physiology, vol. xiv., p. 501.

Gastric digestion is to be considered rather as a preliminary step in
proteolysis, preparatory to the more profound changes characteristic
of pancreatic digestion, in which the ferment trypsin is the important
factor. We can thus see how, as in the case of Czerny’s dogs, an
animal may be perfectly nourished without a stomach, digestive
proteolysis being carried on solely by the pancreatic fluid. You will
remember that two of the dogs operated on by Czerny and his pupils
lived between four and five years after the operation, with the stomach
completely removed, and yet during this period they were well nourished
and ate all varieties of food with apparently a normal appetite.[147]
Evidently, then, in some cases at least, digestive proteolysis
can be carried on without this preliminary action of the gastric
juice. Ogata[148] arrived at essentially the same conclusion by the
establishment of a duodenal fistula, shutting off the stomach from the
intestine by means of a small rubber ball which could be inflated with
water. On then introducing coagulated egg-albumin and other forms of
proteid matter into the duodenum, he found that digestion was at least
sufficiently complete to satisfy all the demands of the system. The
only unsatisfactory result was with collagenous foods, which plainly
showed the need of a preliminary acid digestion. More recently still,
Cawallo and Pachon,[149] working in Richet’s laboratory, have studied
the digestibility of different kinds of proteid foods in a dog, upon
which they had performed a gastrectomy; the entire fundus, as well as
the pyloric portion, of the stomach having been removed. In an animal
so operated upon, after recovery was complete, solid food, as meat,
was completely digested when taken in small quantities at a time. Raw
meat, however, was less completely utilized, the fæces showing portions
of undigested fibres. Still, it was apparent that intestinal digestion
alone was capable of accomplishing all that was necessary for the
complete nourishment of the animal, when it had once become accustomed
to the changed condition of its alimentary tract.

[147] Bunge’s Physiologische und Pathologische Chemie, p. 152.

[148] Ueber die Verdauung nach der Ausschaltung des Magens. Du
Bois-Reymond’s Archiv f. Physiol, 1883, p. 89.

[149] Une observation de chien sans estomac. Comptes Rendus hebd. de la
Société de Biologie, December 1, 1893.

These facts are cited not to belittle the importance of gastric
digestion in the nutrition of the body, but rather to emphasize the
probability that pepsin-proteolysis is simply a preliminary step in
digestion; that its function is not in the direction of a complete
peptonization of the proteid foods ingested, but that its action is
especially directed to the production of soluble products, proteoses,
which can be further digested in the small intestine, or perhaps
directly absorbed after they have passed through the pylorus, or even
from the stomach itself to a certain extent.


SOME PHYSIOLOGICAL PROPERTIES OF PROTEOSES AND PEPTONES.

It is very evident from what has been said that all forms of proteid
matter, _i.e._, all the members of the three main groups spoken
of in our classification of the proteids, excepting only nuclein,
reticulin, and the keratins, are capable of undergoing proteolysis with
pepsin-hydrochloric acid. Further, in every case the main products
of the transformation are proteoses; viz., albumoses, caseoses,
gelatoses, vitelloses, myosinoses, etc., according to the nature of
the proteid undergoing proteolysis; true peptones being formed in
less abundance. Corresponding to each of these groups are primary and
secondary proteoses, all possessed of many points in common, both
chemical and physiological, yet differing from each other in many minor
respects. These are the important products of gastric digestion, of
pepsin-proteolysis, and it may be well to consider for a moment some
of the physiological properties of the proteoses and of peptones as
well, in order that we may the better comprehend the general nature of
these substances with reference to their possible action in the economy.

As far back as 1880, Schmidt-Mülheim[150] discovered that the injection
of aqueous solutions of peptone into the blood-vessels of living dogs
was attended by a series of remarkable phenomena. Thus, the animal
passed at once into a condition of narcosis resembling that produced by
chloroform, accompanied by a fall of general blood-pressure so great
that the animal was liable to die, as from asphyxia. Further, there
was evidence of some marked change in the condition of the blood, as
indicated by loss of the power of spontaneous coagulation, while the
peptone itself evidently underwent some alteration, or else was rapidly
eliminated, since it could not be detected in the blood a short time
after its introduction. These experiments, however, were not conducted
with true peptone but with Witte’s “peptonum siccum,” which at that
time, at least, was composed in great part of proteoses. The general
character of these interesting results was confirmed by Fano,[151] who
found that the injection of so-called peptone in the proportion of 0.3
gramme per kilo. of body-weight was sufficient to bring about complete
narcosis, together with loss of coagulability on the part of the blood.
Very suggestive, however, was the fact that Fano, on trying similar
experiments with the peptone formed by pancreatic digestion, viz.,
with antipeptone, which presumably contained a far smaller proportion
of proteoses, failed to obtain like results; the tryptone, so-called,
being exceedingly irregular in its action, in many cases producing no
effect whatever.

[150] Beiträge zur Kenntniss des Peptons und seiner physiologischen
Bedeutung. Du Bois-Reymond’s Archiv f. Physiol., 1880, p. 33.

[151] Das Verhalten des Peptons und Tryptons gegen Blut und Lymphe.
Ibid., 1881, p. 277.

The discovery at this date of the several albumoses, and their presence
in large amounts in all so-called peptones, led to a study of their
physiological action with special reference to the observations
of Schmidt-Mülheim and Fano. Politzer,[152] working under Kühne’s
guidance, was the first to experiment in this direction, and his
results are full of interest as throwing light on the action of the
individual albumoses. Thus proto, hetero, and deuteroalbumose are all
active physiologically, giving rise when injected into the veins of
dogs and cats to strong narcotic action, varying somewhat in intensity
in different individuals. There is also produced a marked fall in
blood-pressure, due apparently to vaso-motor paralysis, the action
being manifested chiefly, if not wholly, on the splanchnic region.
Thus, after an injection of one of these albumoses, the mesenteric
vessels are always strongly congested, accompanied frequently by the
appearance of a bloody serum in the peritoneal cavity. Narcotic action
is manifested only so long as the blood-pressure remains sub-normal,
and is due presumably to this marked accumulation of blood in the
large abdominal veins, thus leading to anæmia of the brain. Albumoses
and peptones injected into the jugular vein likewise produce fever,
presumably through some action on the nervous system by which the
equilibrium of tissue-metamorphosis is interfered with.[153]

[152] On the Physiological Action of Peptones and Albumoses. Journal of
Physiol., vol. 7, p. 283.

[153] Ott and Collmar: Pyrexial Agents, Albumose, Peptone, and Neurin.
Journal of Physiol., vol. 8, p. 218.

Further, Politzer found that all of the albumoses either delayed or
prevented altogether the coagulation of the blood, in conformity with
the observations of Schmidt-Mülheim and Fano. In all of these actions
the primary albumoses appeared most effective, deuteroalbumose least
so. Heteroalbumose, however, was constantly most active, especially in
delaying the coagulation of the blood. With amphopeptone, there was
far less narcosis and less diminution of blood-pressure, while the
effect on the coagulability of the blood was more or less variable,
frequently being entirely negative. Antipeptone, on the other hand, was
found almost wholly wanting in any constant effects, although in one
instance deep narcosis was produced. Thus, from Politzer’s experiments,
it was made clear that the albumoses, when introduced directly into
the blood-current, possess a far greater toxic action than either
amphopeptone or antipeptone. Albumoses, in sufficiently large doses,
were invariably fatal, while peptones never produced fatal results
so long as the kidneys of the animal remained intact. The extreme
solubility and diffusibility of peptones, coupled perhaps with their
marked diuretic action, lead to rapid elimination through the kidneys,
and their consequent removal from the system.

Many of these observations made with the albumoses I have repeated
with several of the proteoses and peptones more recently studied, as
protocaseose, protoelastose, the globuloses, and others. The results
may be taken as practically confirmatory of the older observations,
and I make mention of them in this general way simply to emphasize
the fact that all of the proteoses, though perhaps showing individual
peculiarities, are possessed of marked physiological properties, which
plainly testify to their toxic nature, when introduced directly into
the blood-current.

Young animals are particularly sensitive to the injection of
proteoses into the blood, even when the introduction takes place very
gradually.[154] Thus, a young, healthy dog of 2 kilos. body-weight,
eight weeks old, died in one hour after the injection into the jugular
vein of 1 gramme of protoalbumose in 20 c.c. of water, thus affording
a good illustration of the extreme toxicity of this albumose when
introduced directly into the blood.

[154] Neumeister: Ueber die Einführung der Albumosen und Peptone in den
Organismus. Zeitschr. f. Biol., Band 24, p. 284.

Of greater interest, physiologically, are the changes the individual
proteoses undergo after their injection into the blood. As already
stated, peptone so injected may appear in the urine wholly unaltered.
Thus, Neumeister[155] has made injections of both amphopeptone
and antipeptone in the case of dogs, and was able to detect the
peptone very quickly in the urine. I have made like experiments with
other forms of peptone and obtained similar results; thus, a pure
amphopeptone formed from casein by pepsin-proteolysis (2 grammes in
15 c.c. water) was injected into the jugular vein of a dog weighing 5
kilos. The urine collected during several hours after the injection
was heated to boiling, and saturated while hot with ammonium sulphate.
The filtrate, on being tested with cupric sulphate and potassium
hydroxide, gave a fairly strong biuret reaction for peptone. Another
similar experiment made with antipeptone, formed from the myosin of
muscle-tissue, gave like results.

[155] Zeitschr. f. Biol., Band 24, p. 287.

With proteoses, however, different results are obtained, as
Neumeister[156] first pointed out. These bodies introduced into the
blood undergo more or less of a change prior to their excretion in
the urine, the change partaking of the character of a hydrolytic
cleavage in which the primary proteoses are transformed into secondary
proteoses, while deuteroproteoses are changed into peptones. This
is not necessarily to be interpreted as meaning that the full
equivalent of the proteose injected appears in the urine, but that the
portion which is eliminated through the kidneys tends to undergo a
transformation somewhere _en route_, akin to the change produced in
pepsin-proteolysis. As to how common or complete this transformation
is under the above circumstances, we have no positive knowledge. Such
a hydrolytic change certainly occurs in the case of the dog, and the
experimental evidence is in favor of the view that the transformation
is effected in the kidneys by the pepsin secreted through the urinary
tubules, where there is momentarily a formation of free acid. In the
rabbit, on the other hand, no such change occurs; the urine from this
animal contains practically no pepsin, and consequently the proteoses
eliminated through the kidneys are excreted unaltered. As, however, the
experiments of Stadelmann[157] and others have shown that the urine of
all carnivora, and of man as well, contains a ferment which, on the
addition of a suitable amount of hydrochloric acid, will digest fibrin
with formation of the ordinary products of pepsin-proteolysis, it is to
be presumed that all proteoses passing through the kidneys will undergo
at least some change prior to their excretion in the urine.

[156] _Ibid._, p. 284.

[157] Untersuchungen über den Pepsin-fermentgehalt des normalen und
pathologischen Harnes. Zeitschr. f. Biol., Band 25, p. 208.

However this may be, it is very evident that the proteoses formed
in gastric digestion cannot be absorbed as such directly into the
blood-current. Introduced into the blood, they behave in such a manner
as to warrant the conclusion that they are truly foreign substances,
and the system makes a brave endeavor to remove them as speedily as
possible. The same may be said of amphopeptones, from which we may
conclude that all of these products of pepsin-proteolysis undergo
some transformation during the process of absorption, by which their
toxicity is destroyed and their nutritive qualities rendered fully
available for the needs of the body. Discussion of this question,
however, will be left until the next lecture.

In view of these pronounced physiological properties of the proteoses,
it is interesting to recall the now well-known fact that many of
the chemical poisons produced by bacteria are proteose-like bodies,
chemically, at least, closely akin to the proteoses resulting from
pepsin-proteolysis. Thus, Wooldridge[158] as early as 1888 pointed out
that an alkaline solution of tissue-fibrinogen exposed to the action
of anthrax-bacilli suffered some change, so that when introduced into
the blood it possessed the power of producing immunity to anthrax.
This observation was verified by Hankin,[159] who further showed that
the substance formed by the anthrax-bacilli was a veritable albumose,
and that it truly possessed the power of producing immunity. Sidney
Martin[160] carried the matter still further, and by growing the
anthrax-bacilli in a pure solution of alkali-albuminate prepared
from blood-serum, proved the formation of both primary and secondary
albumoses, as well as of peptone, leucin, tyrosin, and a peculiar
alkaloidal substance of pronounced toxic properties. Martin finds that
the albumoses are not as poisonous as the alkaloid, and surmises that
the alkaloid is contained in the albumose molecule in the nascent
state; further, he suggests that the albumoses in small doses may exert
some protective influence, while in larger doses they act as vigorous
poisons. How true this may be I cannot say, but my own experience
convinces me that the anthrax-bacilli grown in a culture medium
composed of alkali-albuminate, prepared from egg-albumin, to which
the necessary inorganic salts and some glycerin have been added, do
give rise to albumoses and peptones which are truly endowed with toxic
properties.

[158] Versuche über Schutzimpfung auf chemischem Wege. Du
Bois-Reymond’s Archiv f. Physiol., 1888, p. 527.

[159] British Med. Journal, October, 1889.

[160] Proceed. Royal Society, 1890, vol. 48, p. 78.

Albumose-like bodies have also been obtained by Brieger and
Fränkel[161] with the bacillus of diphtheria. These, too, were endowed
with powerful poisonous properties, and when introduced into the
tissues of the body gave rise to reactions resembling those produced
by the Löffler bacillus. In my own laboratory, recent experiments
made with the bacillus of glanders have shown that when grown in a
slightly acid medium containing alkali-albuminate, albumoses, peptones,
and crystalline bodies such as leucin and tyrosin are formed in
considerable quantities. Kresling[162] has reported similar results.
With the tubercle-bacilli, many like results have been recorded. Thus,
among others, Crookshank and Herroun[163] have reported the finding of
albumoses, peptone, and a ptomaine when the bacilli have been grown in
glycerin agar-agar, and also in fluid media.

[161] Untersuchungen über Bacteriengifte. Berlin, klin. Wochenschrift,
1890, p. 241 and 268.

[162] Ueber die Bereitung des Malleins und seine Bestandtheile.
Abstract in Jahresbericht f. Thierchemie, Band 22, p. 634.

[163] Journal of Physiology, vol. 12, p. 9.

Koch[164] has made a special study of the albumose which he considers
as the specific toxic agent of the so-called tuberculin. This albumose
was found by Brieger and Proskauer[165] to have a somewhat peculiar
composition, inasmuch as it contains forty-seven to forty-eight per
cent. of carbon and only 14.73 per cent. of nitrogen, agreeing,
however, in this respect very closely with the peptone formed from
egg-albumin by the action of bromelin.[166] Still more recently,
Kühne[167] has made a thorough study of this albumose, as well as of
the other products elaborated by the growth of the tubercle-bacillus.
He designates all of the peculiar albumoses formed by these bacilli
as _acrooalbumoses_. They are endowed with marked chemical and
physiological properties, causing a rise of temperature when injected
into the blood, as well as other phenomena more or less pronounced.
It is thus evident there is ample ground for the statement that all
nutritive media in which pathogenic bacteria have been planted are
liable to contain, sooner or later, toxic substances, many of which at
least are closely related to, if not identical with, the albumoses. It
is not my purpose, however, to consider these points in detail, nor to
quote the many results obtained by other workers in this direction.

[164] Deutsche med. Wochenschrift, 1891, p. 1180.

[165] _Ibid._

[166] Chittenden: On the Proteolytic Action of Bromelin, the Ferment of
Pineapple Juice. Journal of Physiology, vol. 13, p. 303.

[167] Weitere Untersuchungen über die Proteine des Tuberculins.
Zeitschr. f. Biol., Band 30, p. 221.

I wish merely to call attention to the fact that the proteoses, and
likewise the peptones formed by pepsin-proteolysis, are more or less
toxic when introduced directly into the blood, and that they share this
property with the proteoses formed by bacterial organisms, or by the
enzymes which they give rise to. In other words, these primary cleavage
or alteration products of the proteid molecule, however produced,
are more or less poisonous, and if introduced into the blood-current
without undergoing previous change may show marked physiological
action. It is, of course, not to be understood that these bodies are
all alike. They are surely closely related and possess many points in
common, especially so far as their chemical properties are concerned,
but their chemical constitution and their physiological action must
vary more or less with their mode of origin.

In any event, it is very evident that the proteoses and peptones
formed in the alimentary tract by pepsin-proteolysis must undergo some
transformation, before reaching the blood-current, by which their
peculiar physiological properties are modified. This modification may
be associated with a conversion into the serum-albumin, or globulin of
the blood. However this may be, the fact remains that these proteoses
formed so abundantly during digestion can be absorbed and serve as
nutriment for the animal body, but between their formation as a result
of proteolysis and their passage into the blood they are exposed to
some agency, or agencies, doubtless in the very act of absorption, by
which a further transformation is accomplished. With this point we
shall be able to deal more in detail in the next lecture.




LECTURE III.

PROTEOLYSIS BY TRYPSIN--ABSORPTION OF THE MAIN PRODUCTS OF PROTEOLYSIS.


PROTEOLYSIS BY TRYPSIN.

In pancreatic digestion, proteids are exposed to the action of an
enzyme of much greater power than pepsin, one endowed with a far
greater range of activity, and consequently proteolysis as it occurs
in the small intestine becomes a broader and more complicated process.
As you well know, the ferment trypsin manifests its power not only in
a more rapid transformation of insoluble proteids into soluble and
diffusible products, but there is a diversity in the character of the
many products formed which testifies to the profound alterations this
ferment is capable of producing. The primary and secondary products of
pepsin-proteolysis, as well as unaltered proteids, are alike subject
to these changes, and bodies of the simplest constitution may result
in both cases from the series of hydrolytic changes set in motion by
this proteolytic enzyme. The power of the ferment as a contact agent is
astonishing, for in the case of trypsin no accessory body is necessary
to bring out its latent power. Water, proteid, and the enzyme at the
body-temperature are all that is necessary to call forth prompt and
energetic hydrolytic action.

Moreover, hydrolysis does not stop with the mere production of soluble
proteoses and peptones, but the hemi-portion of the latter is quickly
broken down into crystalline bodies, such as leucin, tyrosin, lysin,
lysatin, etc. This special characteristic of the ferment testifies
in no uncertain manner to the existence of inherent qualities in
the inner structure of the enzyme peculiar to the body itself. In
general properties and reactions, pepsin and trypsin may be closely
related; both are products of the katabolic action of specific
protoplasmic cells, but the inner nature or structure of the two must
be quite different. Pepsin, as we have seen, is powerless to produce
any change in proteid bodies unless acids are present to lend their
aid. Furthermore, pepsin is limited in its action to the production
of proteoses and peptones, while trypsin gives rise to a series
of hydrolytic cleavages which result in the ultimate formation of
comparatively simple bodies.

Trypsin, however, in its natural environment is dissolved in an
alkaline medium. Its proteolytic action is therefore carried on, under
normal circumstances, in an alkaline-reacting fluid containing 0.5
to 1 per cent. sodium carbonate, and the proteolytic power of the
ferment is unquestionably manifested to the best advantage in such a
medium. At the same time, it will act, and act vigorously, in a neutral
fluid, and likewise in a fluid having a weak acid reaction, provided
there is little or no free acid present. Thus, in experiments[168] on
blood-fibrin it was found that, while a solution of trypsin containing
0.5 per cent. sodium carbonate, digested or dissolved 89 per cent.
of the proteid in three to four hours at 40° C., a perfectly neutral
solution of the ferment, otherwise under exactly the same conditions,
digested 76 per cent., and a 0.1 per cent. salicylic acid-solution of
the enzyme converted 43 per cent. of the proteid into soluble products.

[168] Chittenden and Cummins: Studies in Physiol. Chem., Yale
University, vol. i., p. 135.

With hydrochloric acid, trypsin is quickly destroyed, unless there
is a large excess of proteid matter present,[169] which obviously
means that the acid in such case exists wholly as combined acid.
Indeed, experiments made in my laboratory have shown that as soon as
free acid, especially hydrochloric acid, is present in a solution
containing trypsin, then proteolytic action is at once stopped. When,
however, acids, especially organic acids, are present in a digestive
mixture containing an excess of proteid matter, so that the solution
contains no free acid (or better, with the proteid matter only
partially saturated with acid) then trypsin will continue to manifest
its peculiar proteolytic power, although to a considerably lessened
extent. Hence, it is evident that the ferment may exert its digestive
power under the three possible sets of conditions which, under varying
circumstances, frequently prevail in the small intestine.

[169] Mays: Untersuchungen aus d. physiol. Institute d. Universität
Heidelberg, Band iii., p. 378; also Langley: On the Destruction of
Ferments in the Alimentary Canal, Journal of Physiology, vol. iii.,
p. 263.

In considering the general phenomena of proteolysis by trypsin, one
is especially impressed by the large and rapid formation of peptone
which almost invariably results from the action of a moderately strong
solution of the ferment, on nearly every form of proteid matter. To
be sure, primary products are first formed, but these are quickly
converted into peptone, and a little experience in studying the
action of pepsin and trypsin soon reveals the fact that the latter is
especially a peptone-forming ferment. In other words, it is peculiarly
adapted to take up the work where it has been left by pepsin and, if
necessary, carry forward the hydrolytic change even to the extent of a
conversion of the entire hemi-moiety into crystalline products.

The primary products of trypsin-proteolysis, however, are not exactly
identical with those formed by pepsin. Thus, protoproteoses and
heteroproteoses seldom appear in an alkaline trypsin digestion; the
proteid matter being in most cases, at least, directly converted into
soluble deuteroproteoses,[170] which are then transformed by the
further action of the ferment into peptones and other products. Hence,
we may express the order of events in the trypsin digestion of a native
proteid as follows:

          Native proteid.
                 |
       Amphodeuteroproteoses.
                 |
           Amphopeptones.
             ╱      ╲
           ╱          ╲
    Antipeptone.   Hemipeptone.
                    ╱   |   ╲
                  ╱     |     ╲
            Leucin.  Tyrosin.  Aspartic acid, etc.

[170] R. Neumeister: zur Kenntniss der Albumosen. Zeitschr. f. Biol.
Band 23, p. 378.

In the digestion of fresh blood-fibrin with trypsin, there is
plainly a preliminary solution of the proteid without any marked
transformation or cleavage occurring, the soluble product being
apparently a globulin, coagulating at about 75° C.,[171] viz., at
approximately the same temperature as serum-globulin. This body,
however, quickly disappears, giving place to true deuteroproteoses as
the ferment-action commences; for it is not probable that this globulin
is a product of enzyme-action, but rather represents a simple solution
of the fibrin by the alkaline fluid and salts. In any event, this
globulin-like substance is not formed in the pancreatic digestion of
coagulated-albumin, serum-albumin, or vitellin, and hence cannot be
considered as a true product of trypsin-proteolysis.

[171] Jac. G. Otto: Beiträge zur Kenntniss der Umwandlung von
Eiweissstoffen durch Pancreas-ferment. Zeitschrift f. physiol. Chem.,
Band 8, p. 129.

The fact that deuteroproteoses are the primary products of
trypsin-digestion again emphasizes the natural adaptability of this
ferment to the part it has to play in the digestive process. Its
natural function is to take up the work where left by pepsin, and
carry it forward to the necessary point; and hence, when acting
upon a native proteid the primary products of its action correspond
to the secondary products of pepsin-proteolysis. Trypsin is thus
equally efficient in the digestion of all native proteids, but the
products of such action are always deuteroproteoses, peptones, and
crystalline amido-acids. It is to be remembered, however, that in
trypsin-proteolysis the deuteroproteoses and the amphopeptones must
necessarily be represented by bodies in which there is a preponderance
of anti-groups. In pepsin-proteolysis, as we have seen, the hemi- and
anti-groups of the proteid molecule remain more or less united, but in
pancreatic digestion, the formation of amphopeptone is quickly followed
by the breaking down of a portion of the hemipeptone into leucin,
tyrosin, etc. thus leaving a larger proportion of the anti-moiety in
the remaining amphopeptone.

Theoretically, at least, in the-case of a vigorous and long-continued
pancreatic digestion, all of the hemipeptone formed from any native
proteid can be converted into crystalline and other products, thus
leaving a true antipeptone resistant to the further action of trypsin.
Hence, we are prone to speak of the peptone of pancreatic digestion
as antipeptone, although, as can be readily seen, the exact nature of
the peptone, _i. e._, the relative proportion of hemi- and anti-groups
it contains, will obviously depend upon the length of the digestion
and the strength of the ferment. Again, it is possible, as certain
facts seem to suggest, that the amido-acids which are so readily
formed from hemipeptone may come in part directly from the hydration
of a portion of the hemideuteroproteose, without passing through the
preliminary stage of hemipeptone. If so, we have another source of
variation in the relative proportion of hemi- and anti-moieties in the
deuteroproteoses and peptones of pancreatic digestion. Still again, it
is to be remembered that in normal digestive proteolysis, as it occurs
in the living intestinal tract, the proteid matter to be acted upon
has already passed through certain preliminary stages in, its transit
through the stomach, as a result of which still further variations in
the proportion of hemi- and anti-groups may be possible.

It is thus plainly evident, in view of the ready cleavage of
the hemi-group into amido-acids, that the primary products of
trypsin-proteolysis, the proteoses and peptones, must necessarily be
composed in great part of those complex and semi-resistant atoms which
we include under the head of the anti-group. However much one may be
skeptical about the real existence of so-called hemi- and anti-groups,
there is no gainsaying the fact that a given weight of native proteid,
like egg-albumin or blood-fibrin, cannot be converted wholly into
crystalline or other simple products by trypsin; indeed, it is quite
significant that at the end of a long-continued treatment with an
alkaline solution of the pancreatic ferment, there is usually found
about fifty per cent, of peptone, while the other fifty per cent. of
the proteid is represented mainly by more soluble products, such as
the amido-acids. It is also significant that the peptone obtained from
an artificial pancreatic digestion, where the proteolytic action has
been long-continued and vigorous, resists the further action of the
ferment. In other words, it is the so-called antipeptone. In line with
this result is the fact that the peptones formed in pepsin-proteolysis,
when treated with an alkaline solution of trypsin, are converted into
amido-acids and other bodies of simple constitution to the extent of
about fifty per cent. This is easily explainable on the ground that
the hemi-portions of the above peptones are broken down into simple
products, while the anti-portions remain unchanged, being resistant to
the ferment and thus leading to a separation of the two groups, or at
least to the isolation of the anti-molecules.

There is much that might be cited in further support of these views,
but doubtless I have said enough to make it plainly evident that in
the pancreatic digestion of any native proteid, not more than one-half
can at the most be transformed into crystalline products, while the
other half will be represented mainly by a peptone incapable of further
change by trypsin. Similarly, the products of pepsin-proteolysis
exposed to the action of trypsin may undergo a like separation, the
hemi-groups only breaking down into simple products. Hence, the whole
theory of the hemi- and anti-moieties of the proteid molecule means
simply that of the many complex atoms composing the molecule, one-half
are easily decomposable by the pancreatic ferment, while the other half
are more resistant and make up the so-called anti-group.

In any active pancreatic digestion of either a native proteid, or of
the products of pepsin-proteolysis, the anti-group is represented
mainly by antipeptone, although there is often found a small amount of
a peculiar antialbumid-like body, insoluble in the weak alkaline fluid.
Antipeptones, thus far studied, when entirely free from proteoses, are
characterized by a low content of carbon, like the amphopeptones from
pepsin-proteolysis. The following table shows the composition of a few
typical examples:


COMPOSITION OF ANTIPEPTONES.

         From       From        From
         blood-     blood-      anti-       From       From
        fibrin.    fibrin.    albumose.    casein.     myosin.
         [172]      [173]       [174]       [175]      [176]

    C    47.30      49.59       48.94       49.94      49.26
    H     6.73       6.92        6.65        6.51       6.87
    N    16.83      15.79       15.89       16.30      16.62
    S     0.73       ---         ---         0.68       1.16
    O    28.41       ---         ---        26.57      26.09

[172] Kühne and Chittenden: Studies in Physiol. Chem. Yale Univer.,
vol. ii., p. 40.

[173] J. Otto: Zeitschr. f. physiol. Chem., Band 8, p. 146.

[174] Kühne and Chittenden: Zeitschr. f. Biol., Band 19, p. 196.

[175] Chittenden: Studies in Physiol. Chem. Yale Univer., vol. iii.,
p. 101.

[176] Chittenden and Goodwin: Journal of Physiol., vol. xii., p. 34.

From these data it is evident that, while each individual peptone may
have a composition peculiar to itself, they are all alike in possessing
a relatively low content of carbon. The antialbumid, however, split off
in these hydrolytic changes, like the antialbumid formed by the action
of dilute acids at 100° C., is characterized by a correspondingly high
content of carbon and a low content of nitrogen. As an illustration,
may be mentioned the myosin-antialbumid formed in the digestion of
myosin from muscle-tissue by an alkaline trypsin-solution. This body
contains 57.48 per cent. of carbon, 7.67 per cent. of hydrogen, 13.94
per cent. of nitrogen, 1.32 per cent. of sulphur, and 19.59 per
cent. of oxygen.[177] It is only necessary to compare these figures
with those expressive of the composition of myosin-antipeptone, to
appreciate how wide a gap there is between these two products of
trypsin-proteolysis, and both members of the anti-group. Antialbumid,
however, is a peculiar product, one which is liable to crop out
somewhat unexpectedly, and with varying shades of resistance toward
the proteolytic ferments. As formed in pepsin-proteolysis, it is more
or less readily soluble in sodium carbonate, and in part readily
convertible into antipeptone by trypsin. Still, the same substance, or
at least a closely related body, makes its appearance in the form of
an insoluble residue whenever a native proteid is digested by trypsin.
At times, the amount of this insoluble product may be quite large,
even reaching to one-fourth of the total proteid matter;[178] but when
so formed in the intestine it must entail a heavy loss of nutriment,
for whenever the anti-group is split off after this fashion it becomes
very resistant to the further action of the ferment. Separating in
this manner from an artificial digestive mixture, it may be dissolved
in dilute caustic alkali, reprecipitated by neutralization, and then
once again brought into solution with dilute sodium carbonate. In this
form, it will yield some antipeptone by the further action of trypsin,
although even then a large amount of the antialbumid is prone to
separate out as a gelatinous coagulum, more or less resistant to the
further action of the ferment.

[177] Chittenden and Goodwin: Journal of Physiol., vol. xii., p. 36.

[178] Kühne und Chittenden: Ueber die nächsten Spaltungsproducte der
Eiweisskörper. Zeitschr. f. Biol., Band 19, p. 196.

The peculiar action of trypsin, however, as a proteolytic enzyme is
shown in the production of a row of crystalline nitrogenous bodies
of simple constitution whenever the ferment is allowed to continue
its action for any length of time, either on native proteids or on
proteolytic products containing the hemi-group. This, to be sure, is a
fact long known, but it gains added significance as year by year new
bodies are discovered as products of trypsin-proteolysis with various
forms of proteid matter. The very character of the bodies originating
in this manner gives evidence of the far-reaching decompositions
involved; decompositions which are perhaps attributable as much to the
innate tendencies of the proteid material as to the specific action of
the ferment. As representatives of this peculiar line of cleavage, we
have first the well-known bodies, leucin and tyrosin; leucin, a body
belonging to the fatty acid series, long known as amido-caproic
acid, but now generally considered as amido-isobutylacetic acid,
(CH_{3})_{2} CHCH_{2} CH(NH_{2}) COOH; and tyrosin, a body
belonging to the aromatic group, having the formula

                 OH
               ╱
    C_{6}H_{4}⧼
               ╲
                 CH_{2} CH(NH_{2}) COOH,

and known as oxyphenyl-amido-propionic acid.

These two bodies are therefore representatives of two distinct groups
or radicals present in the hemi-portion of the proteid molecule; the
first belonging to the fatty acid series, the second to the aromatic
group from which come such well-known bodies as indol, skatol, benzoic
acid, and other substances prominent in proteid metabolism. Moreover,
these two hydrolytic products of trypsin-proteolysis are formed in
considerable quantity, at least in an artificial digestion. Thus,
Kühne has reported the finding of 9.1 per cent. of leucin and 3.8 per
cent. of tyrosin as the result of a typical digestion, and I have
tried many similar experiments with like results. Further, we know
from observations made by different investigators that both leucin and
tyrosin may be formed in considerable quantities in trypsin-proteolysis
as it occurs in the living intestine. But to this point we shall return
later on.

Besides leucin and tyrosin, aspartic acid and glutamic acid have long
been known as decomposition-products of the vegetable proteids. Thus,
both acids were discovered by Ritthausen and Kreusler[179] in the
cleavage of such proteids by boiling dilute acid. Hlasiwetz and
Habermann[180] likewise obtained aspartic acid in large quantity by
the breaking down of animal proteids under the influence of bromine.
Further, Siegfried[181] has recently obtained glutamic acid as a
product of the decomposition of the phosphorus-containing proteid,
reticulin, from adenoid tissue. As products of trypsin-proteolysis,
Salkowski and Radziejewski[182] found aspartic acid in the digestion
of blood-fibrin; and v. Knieriem[183] likewise obtained it in the
digestion of gluten from wheat. Both of these acids belong to the
fatty acid series, the aspartic acid being a dibasic acid,
COOH. CH_{2}CH(NH_{2}). COOH, or amido-succinic acid, while glutamic
acid, COOH. C_{3}H_{5}(NH_{2}). COOH, is likewise a dibasic acid,
known as amido-pyrotartaric acid.

[179] Verbreitung der Asparaginsäure und Glutaminsäure unter den
Zersetzungs-producten der Proteinstoffe. Journal f. prakt. Chemie, Band
3, p. 314.

[180] Ueber die Proteinstoffe. Liebig’s Annalen, Band 159, p. 304.

[181] Ueber die chemischen Eigenschaften des Reticulirten Gewebes.
Habilitationschrift. Leipzig, 1892.

[182] Bildung von Asparaginsäure bei der Pancreas-Verdauung. Bericht.
d. Deutsch. chem. Gesellsch., Band 7, p. 1050.

[183] Asparaginsäure, ein Product der künstlichen Verdauung von Kleber
durch die Pancreas-Drüse. Zeitschr. f. Biol., Band 11, p. 198.

Of more interest physiologically, are the recently discovered
nitrogenous bases lysin and lysatinin, or lysatin. These two bodies
were first identified by Drechsel[184] and his co-workers as products
of the decomposition of various proteids, when the latter are boiled
with hydrochloric acid and stannous chloride. They were first obtained
by Drechsel as cleavage products of casein.[185] Later, Ernst
Fischer,[186] working under Drechsel’s direction, separated them as
decomposition-products of gelatin; while Siegfried[187] obtained them
as products of the cleavage of conglutin, gluten-fibrin, hemiprotein,
and egg-albumin, by boiling with hydrochloric acid and stannous
chloride. In all of these cases it is obvious, from the method of
treatment pursued, that the two bodies result from a simple hydrolytic
cleavage of the proteid molecule. Hence, it might be assumed that
these two bases would likewise be formed in trypsin-proteolysis. This
assumption, Hedin,[188] working in Drechsel’s laboratory, has proved to
be correct, and furthermore he has shown that the amount of these bases
formed in pancreatic digestion is not inconsiderable. Thus, as products
of the digestion of three kilos. of moist blood-fibrin with an alkaline
solution of trypsin, 28 grammes of pure platino-chloride of lysin were
obtained, and sufficient lysatinin to establish its identity.

[184] Der Abbau der Eiweissstoffe. Du Bois-Reymond’s Archiv. f.
Physiol., p. 248. 1891.

[185] Zur Kenntniss der Spaltungsproducte des Caseins. _Ibid._, p. 254.
1891.

[186] Ueber neue Spaltungsproducte des Leimes. _Ibid._, p. 265. 1891.

[187] Zur Kenntniss der Spaltungsproducte der Eiweisskörper. _Ibid._,
p. 270. 1891.

[188] Zur Kenntniss der Producte der tryptischen Verdauung des Fibrins.
_Ibid._, p. 273. 1891.

Lysin has the composition of C_{6} H_{14} N_{2} O_{2}, being a
diamido-caproic acid, a homologue of diamido-valerianic acid. Hence,
this body, like leucin or amido-caproic acid, is a representative of
the fatty acid group, the chemical relationship between the two bodies
being plainly apparent from their constitution. The constitution
of lysatinin is less definitely settled, but apparently it has the
composition of a creatin, its formula being C_{6}H_{13}N_{3}O_{2}, in
which case it might be more appropriately termed lysatin. The special
point of interest, however, connected with this latter body as a
product of trypsin-proteolysis is the fact that by simple hydrolytic
decomposition, all chance of oxidation being excluded, it can break
down into urea.[189] For years, chemists have been seeking to trace
out the line of cleavage or decomposition by which urea results in
proteid metabolism. In the nutritional changes of the body, nearly all
the nitrogen of the ingested proteid food is excreted in the form of
urea, but chemists working with dead food-albumin have been heretofore
unable to break down proteid matter directly into urea. This, however,
Drechsel has now succeeded in doing, and it is to be especially noted
that the line of decomposition or cleavage is simply one of hydration,
in which the proteid molecule, either through the action of boiling
dilute acids, or through the more subtle influence of the hydrolytic
enzyme, trypsin, is gradually broken down into cleavage products, from
one or more of which comes lysatin. The very resemblance of this body
to creatin suggested that, since the latter breaks down into urea and
sarcosin when boiled with baryta water, lysatin might possibly behave
in a similar manner. This, as has been previously stated, was found to
be the case, and Drechsel obtained from ten grammes of a double salt of
lysatin and silver one gramme of urea nitrate, by simple boiling with
baryta water.

[189] Drechsel: Ueber die Bildung von Harnstoff aus Eiweiss. Du
Bois-Reymond’s Archiv f. Physiol., p. 261. 1891.

It is thus evident that a certain amount of urea may come from the more
or less direct hydrolysis of proteid matter in the intestinal canal,
all but the last steps in the process being the result of the ordinary
cleavage processes incidental to trypsin-proteolysis. This fact affords
additional evidence of the profound changes set in motion by this
proteolytic enzyme. It is not, of course, to be understood that all the
urea formed in the body has its origin in this manner. Such a method of
decomposition taking place in the intestinal tract would be exceedingly
unphysiological and wasteful, but we can readily see how such a line of
cleavage might result in inestimable gain to the economy in cases where
excess of proteid food has been ingested. Under such circumstances, a
portion of the surplus might be broken down directly in the intestine
into this urea-antecedent, and thus quickly removed from the system
with a minimum amount of effort on the part of the economy. Drechsel
estimates that about one-ninth of the urea daily excreted may come from
the direct decomposition of lysatin, the latter obviously having its
origin in trypsin-proteolysis.

Another product of trypsin-proteolysis which has long been recognized,
although its real nature has not been known, is tryptophan or
proteinochromogen. This body is not only a product of the pancreatic
digestion of proteids, but it is also formed whenever native proteids
are broken down through any influence whatever, the substance coming
presumably from the hemi-moiety of the molecule. It is especially
characterized by the bright- compound it forms with either
chlorine or bromine, so that for a long time it went by the mystical
name of the “bromine body.” When brought in contact with either of
these agents, it immediately combines with them to form a new compound
of an intense violet color, termed proteinochrome. This constitutes
the usual test for its presence, a little bromine water, for example,
quickly bringing out a violet color when added to a fluid containing
the chromogen. The body is readily soluble in alcohol, and hence can
be easily separated from the primary products of trypsin-proteolysis,
such as the proteoses and peptones. Krukenberg considered the substance
not a true proteid, but rather a body belonging to the indigo-group;
but Stadelmann, who has given the matter a very thorough investigation,
comes to the conclusion that it is truly a proteid body, in part
closely related to peptone, although in many ways quite different.

The following composition of bromine proteinochrome, as determined by
Stadelmann,[190] shows the general nature of the compound formed when
bromine combines with the chromogen:

           _A_      _D_
    C     49.00    48.12
    H      5.28     5.09
    N     10.99    11.92
    S      3.77     3.10
    O     11.01    12.00
    Br    19.95    19.77

[190] Ueber das beim tiefen Zerfall der Eiweisskörper entstehende
Proteinochromogen, den die Bromreaction gebenden Körper. Zeitschr. f.
Biol., Band 26, p. 521.

From the average of the several results obtained, it would appear
that the proteinochromogen, which could not be isolated by itself
in sufficient purity for analysis, must contain approximately 61.02
per cent. of carbon, 6.89 per cent. of hydrogen, 13.68 per cent. of
nitrogen, 4.69 per cent. of sulphur, and 13.71 per cent. of oxygen.
As a proteid-like body, it is thus especially characterized by an
exceedingly high content of carbon and a high content of sulphur. As
a product of trypsin-proteolysis, it must presumably come from the
cleavage of hemipeptone, which, however, contains only 0.75 per cent.
of sulphur. But as we have seen, this latter body breaks down by
further cleavage into substances such as leucin, tyrosin, lysin, etc.,
which contain no sulphur whatever, and as there is no elimination
of sulphur in this process through formation of hydrogen sulphide
gas or otherwise (putrefaction being excluded by the presence of
either chloroform or thymol), it follows that this surplus sulphur
must accumulate somewhere. The high content of carbon, however, in
proteinochromogen is sufficient evidence that the substance cannot
have its origin in a simple cleavage of hemipeptone. On the other
hand, everything points to a synthetical process, in which two or more
cleavage products of the proteid molecule combine and form a new body,
such as proteinochromogen, containing all the sulphur cast off from the
hemipeptone in the production of the crystalline bodies, and having in
itself properties common to peptone and to a body of the indigo-group,
the latter obviously coming from some aromatic antecedent.

In view of the apparent complexity of the processes attending
trypsin-proteolysis, it is not strange that even simpler substances
than those already described should make their appearance. Thus, when
it was suggested that ammonia, NH_{3}, might be formed under the
influence of trypsin, it was not considered at all improbable, for in
the hydrolytic decomposition of proteids by boiling dilute acid, as
well as by baryta water, it had long been known as a prominent product.
Obviously, in trypsin-proteolysis, the one thing to be guarded against
in proving the formation of ammonia is the contaminating influence
of bacteria. Hirschler,[191] however, with a full recognition of this
danger, made digestions of blood-fibrin with trypsin extending only
through four hours and at a temperature of 32° C., and yet he obtained
plain evidence of the formation of ammonia. Stadelmann,[192] with
still greater precautions to exclude all bacterial agencies, using
boiled fibrin as the material to be digested and thymol to prevent
any possible infection of the digestive mixture, proved conclusively
that ammonia was formed as a result of trypsin-proteolysis. Thus, in
the digestion of 35 grammes of boiled blood-fibrin with 60 c. c. of
a pancreas infusion for three days, 20.8 milligrammes of NH_{3} were
developed, presumably coming from the liberation of a certain amount
of nitrogen attendant upon the formation of such bodies as leucin and
tyrosin, which contain considerably less nitrogen than their direct
antecedent hemipeptone, or the original proteid. We thus have striking
proof of the ability of this peculiar proteolytic enzyme to set in
motion hydrolytic changes which may extend even to the production of
such simple substances as ammonia, thus making still more striking
the parallelism between trypsin-proteolysis on the one hand, and the
artificial hydrolysis produced by boiling dilute acids on the other.

[191] Bildung von Ammoniak bei der Pancreasverdauung von Fibrin.
Zeitschr. f. physiol. Chem., Band 10, p. 302.

[192] Bildung von Ammoniak bei Pancreasverdauung von Fibrin. Zeitschr.
f. Biol., Band 24, p. 261.

In view of all these facts regarding the nature of the products
obtainable by pancreatic proteolysis, it is very evident that many
chemical changes may take place side by side in a vigorous pancreatic
digestion of proteid matter. We know without a shadow of doubt that all
of the bodies enumerated as products of pancreatic digestion are the
results of trypsin-proteolysis, and not the products of putrefactive
changes. Bacteria, it is true, are able to produce many like products,
and in the living intestinal tract exercise an important influence,
especially in the breaking down of resistant forms of proteid matter,
and in the decomposition of surplus material which has escaped the
pancreatic ferment. But all the bodies described above are readily
obtainable by trypsin-proteolysis under conditions which exclude all
possibility of bacterial action.

Granting, then, as we must, that these various bodies are all products
of pancreatic proteolysis when the process is carried on in beakers
or flasks, we need to consider next how far such bodies appear in
the natural process as it takes place in the living intestine. We
know indeed that the natural and the artificial processes are very
much alike so far as the qualitative results are concerned, but what
differences there may be between the quantitative relationships in the
two cases is less certain. One might naturally reason that, with the
facilities for rapid absorption that exist in the small intestine,
trypsin-proteolysis would rarely proceed beyond the peptone stage,
yet we have ample evidence that, under some circumstances at least,
both leucin and tyrosin are formed in considerable quantities in the
intestine.

It obviously makes a very great difference to the economy in what
form the proteid matter ingested leaves the intestine on its way into
the blood-current. It has been more or less generally assumed that,
under the ordinary circumstances existent in the intestinal tract, the
crystalline and other bodies coming from the more profound changes
incidental to trypsin-digestion are rarely formed, mainly on the
ground that such transformations would entail great loss of nutritive
material to the blood. Years ago, Schmidt-Mülheim[193] made a series
of experiments on the changes which proteid foods undergo in different
portions of the alimentary tract, from which he concluded that leucin
and tyrosin are formed in such small quantities in natural pancreatic
digestion that they represent only a very small part of the nitrogen
absorbed from the intestine. This conclusion has been more or less
generally accepted, especially as several observers have reported
finding only small amounts of these bodies in the intestine under what
might be assumed to be favorable circumstances for their formation. In
artificial digestions, on the other hand, as we have seen, leucin and
tyrosin, together with the other simple bodies described, may appear
in large quantities. Obviously, two suggestions present themselves as
explanatory of this difference; either there is such a rapid absorption
of these crystalline products from the intestine that they cannot be
detected other than as mere traces, or else the natural process takes
a different course from the artificial, owing to the rapid withdrawal
from the intestine of the antecedent of the leucin and tyrosin, viz.,
the hemipeptone.

[193] Untersuchungen über die Verdauung der Eiweisskörper. Du
Bois-Reymond’s Archiv f. Physiol., 1879, p. 39.

Concerning this point, Lea[194] has recently reported some experimental
evidence obtained by a comparative study of artificial pancreatic
digestion as carried on in a flask, with similar digestions carried on
in parchment dialyzer tubes, the latter so arranged that the diffusible
products of proteolysis can pass from the tube into the surrounding
fluid. As Lea justly says, this whole question of the formation
of leucin by proteolysis is a very important one, since it bears
closely upon one of the possible methods by which urea may be quickly
formed from proteid food. Thus, we have evidence that when leucin is
administered to mammals a portion of its nitrogen, at least, quickly
reappears as urea and uric acid in the urine.[195] Further, there is
a certain amount of evidence that this transformation takes place in
the liver, viz., in the organ where leucin absorbed from the intestine
would naturally be first carried.[196]

[194] A Comparative Study of Artificial and Natural Digestions. Journal
of Physiology, vol. xi, p. 226.

[195] E. Salkowski: Weitere Beiträge zur Theorie der Harnstoffbildung.
Zeitschr. f. Physiol. Chem., Band 4, pp. 55 and 100.

[196] W. Salomon: Ueber die Vertheilung der Ammoniaksalze im
thierischen Organismus und über den Ort der Harnstoffbildung. Virchow’s
Archiv, Band 97, p. 149.

Obviously, the main point to be gained in a dialyzer-experiment is
the removal of the soluble products of digestion as soon as they are
formed; but peptones are not rapidly diffusible, and the process,
as noted under the head of gastric digestion, cannot be considered
in any sense as yielding the same results as might be obtained in
the living intestine. Still, the method offers a closer approach
to the natural process than when carried on in a flask, and the
results are of interest. Thus, Lea finds in the first place that
in a dialyzer-digestion the proteid is more quickly dissolved, and
that there is far less tendency for the formation of an insoluble
antialbumid with its natural resistance to the ferment. Still, it is
to be noticed that the amount of this antialbumid-residue formed by
trypsin-proteolysis in a flask is mainly dependent upon the strength
of the ferment solution, and the character of the proteid undergoing
digestion. If the latter is in a fairly digestible form, and the enzyme
solution reasonably active, then even the flask-digestion may show
almost no residue of antialbumid. Yet there is at least a shade of
difference in the two cases, which may be expressed by the statement
that trypsin-proteolysis, as carried on in a dialyzer-tube, is prone
to give less insoluble antialbumid than a corresponding digestion
in a flask. Further, the amount of leucin and tyrosin formed in a
flask-digestion is always greater than in a dialyzer-digestion, other
conditions being equal. Naturally, these results help us very little
in drawing any conclusions regarding the extent to which leucin and
tyrosin may be formed in the intestine. They merely emphasize the
fact that the withdrawal of a certain quantity of hemipeptone from
the digestive mixture tends to reduce by so much the yield of leucin
and tyrosin. It is hardly to be assumed, however, that the rate of
withdrawal of peptone from the intestine can keep pace with its
formation, especially when it is remembered that the proteid matter
coming into the small intestine, owing to its preliminary treatment
in the stomach, is in a comparatively digestible condition. Further,
the pancreatic juice is a remarkably active fluid, and proteolysis
under its influence must make rapid strides. I can easily conceive
that proteolysis by trypsin, when carried on in a flask, may lead to
the formation of much larger amounts of leucin and tyrosin, and of
other bodies as well, than occurs in the natural process; but there is
certainly no ground for the belief that leucin and tyrosin are wholly
wanting in pancreatic proteolysis as it occurs in the intestine.

With a view to obtaining some positive evidence on this point I have
tried a few experiments on animals, the results of which have convinced
me that, in the case of dogs, at least, both leucin and tyrosin may
be formed in natural pancreatic digestion in considerable quantities.
Thus, in one experiment a good sized dog, kept without food for two
days, was fed four hundred grammes of chopped lean beef at 8 A.M.
At 2 P.M. the animal was killed and the intestine ligatured close
to the pylorus. The lower end of the small intestine was likewise
ligatured. The portion inclosed between the two ligatures was then
removed from the body, and the contents of the intestine pressed and
rinsed out with distilled water. In the stomach, was found a small
amount of semi-digested matter weighing about fifty grammes. The
material obtained from the intestine was strained through mull, the
fluid rendered faintly acid with acetic acid, and heated to boiling.
The clear filtrate from this precipitate was concentrated to a very
small volume, and while still hot precipitated with a large amount of
ninety-five per cent. alcohol. A small gummy precipitate resulted,
which was thoroughly extracted with boiling alcohol and the washings
added to the alcoholic filtrate. The precipitate contained some
deuteroproteose and a small amount of true peptone.

The alcoholic fluids were evaporated to a small volume and set aside
in a cool place. As a result, quite a separation of leucin and tyrosin
occurred in the characteristic crystalline forms. No attempt was
made to effect a quantitative separation of the two bodies, but the
mixed precipitate finally obtained weighed, after recrystallization,
over three-fourths of a gramme. Leucin was plainly in excess, but
considerable tyrosin must have been left in the alcoholic precipitate,
owing to its greater insolubility in this menstruum. This experiment
is almost a counterpart of one reported by Lea,[197] and like
his indicates that both leucin and tyrosin may be formed in not
inconsiderable quantities by pancreatic proteolysis as it occurs in
the intestine. This being so, one is naturally called on to explain
“the physiological significance of a process which at first sight
appears to result in a degradation of the potential energy of proteids,
under conditions such that the energy set free can be of little use
to the economy.”[198] But it is quite possible, as Lea has suggested,
that these amido-bodies have an important part to play in some of
the synthetical or other processes of the organism, and that their
formation is consequently necessary for the well-being of the body.
Whether this is so or not, we may well consider the formation of these
amido-acids in pancreatic proteolysis as a means of quickly ridding the
body of any excess of ingested proteid food, with the least possible
expenditure of energy on the part of the system. This has always seemed
to me the probable purpose of the profound changes which the pancreatic
ferment is capable of inducing.

[197] Journal of Physiology, vol. xi, p. 255.

[198] Lea: loc. cit.

The primary object of both gastric and pancreatic proteolysis is to
render the proteid foods more easily available for the needs of the
economy, viz., to aid in their absorption and consequent distribution
to the master tissues and organs of the body. This is doubtless
fully accomplished by the formation of the so-called primary and
secondary products of proteolysis, _i. e._, the proteoses and peptones
which are, comparatively, not far removed from the mother-proteid,
except in solubility and other minor points. In the ferment trypsin,
however, we have a special agent endowed with the power of carrying
on the hydrolytic cleavage to a point where exceedingly simple bodies
result, and through whose agency any excess of proteid material in the
intestinal canal may be quickly broken down into a row of products
easily removed from the system. It is to be remembered, however, that
the very nature of the proteid molecule precludes the possibility
of anything like a complete decomposition into crystalline or other
simple products. Full fifty per cent. of the peptone formed must
be antipeptone, which cannot be further changed by trypsin under
any circumstances, so that, whether the amount of proteid in the
intestine be large or small, or whether it is exposed for a longer or
shorter period to trypsin-proteolysis, there will always be a fairly
large amount for absorption. This may well be considered as one of
the reasons for the peculiar structure of the proteid molecule, the
anti-group being always available for the direct nutrition of the body,
while the representatives of the hemi-group, especially when proteid is
present in excess, can be quickly and readily broken down into simple
products. In other words, the direct formation of these simple bodies
in the intestine furnishes a short path to urea, thus leading to the
rapid elimination of any excess of proteid material.

We may well attribute to the epithelial cells of the intestine the
power, under normal circumstances, of regulating and controlling, even
though indirectly, the order of events in the intestine. Just as the
so-called secreting cells of the _tubuli uriniferi_ may lose for a time
their power to pick out from the blood material destined for the urine,
being clogged or exhausted by continued effort, so the epithelial cells
of the intestine, which play such an important part in the absorption
of proteid matters from the alimentary tract, may, in the presence
of an excess of proteid matter, become temporarily exhausted, and,
refusing passage to the proteoses and peptone formed by proteolysis,
render possible further hydrolytic cleavage into leucin, tyrosin,
lysatin, etc.; bodies which, by one method or another, can be readily
transformed into urea. At the same time, as already stated, it seems
more than probable that some formation of these amido-acids always
occurs in the intestine, and that these bodies have some specific part
to play in the normal processes of metabolism going on in the body.
The more one studies the processes of nutrition in general, the more
one is impressed with the view that there is a purpose in everything,
and that the formation of even such bodies as leucin and tyrosin may
be connected with hidden processes, the key to which has not yet
been found. We see an analogous case, perhaps, in the action of the
inorganic salts in nutrition, some of which, at least, neither undergo
change themselves nor induce changes in other substances, and yet we
know their presence is indispensable for keeping up the normal rhythm
of the nutritional processes of the body.


ABSORPTION OF THE MAIN PRODUCTS OF PROTEOLYSIS.

In ordinary proteolytic action, both in the stomach and intestine,
it is very apparent that the primary products of proteolysis, the
proteoses and peptones, are the chief products formed, and that under
normal circumstances the greater portion of the proteid food finds its
way from the alimentary canal into the blood, after transformation
into one or more of these two classes of products. At the same
time, it must be borne in mind that even the acid-albumin formed by
pepsin-hydrochloric acid may be absorbed without undergoing further
change. The view once held, that the rate of absorption from the
alimentary tract stands in close relation to the diffusibility of the
products formed, and that non-diffusible substances are incapable of
absorption, is no longer tenable. Absorption from the intestine is to
be considered rather as a process involving the vital activity of the
epithelial cells of the intestinal mucous membrane, where chemical
affinities and other like factors play an important part in determining
the rate and order of transference through the intestinal walls into
the blood and lymph. Thus, we have abundant evidence that native
proteids which have not undergone proteolysis may be absorbed from
the intestine, at least to a certain extent, provided they have been
dissolved; _i. e._, converted into acid-albumin, or alkali-albuminate,
by the gastric or pancreatic juice. We have a practical demonstration
of this possibility in the early experiments of Voit and Bauer,[199] as
well as in many later ones that need not be mentioned here. Further,
the recent experiments of Huber[200] have given us quantitative data
on the rate of absorption of fluid egg-albumin when introduced into
the large intestine in the form of a clyster, showing that even fairly
large amounts of a natural proteid may be absorbed without undergoing
proteolysis if mixed with a neutral salt, like sodium chloride. To be
sure, the rate of absorption is greatly increased when the albumin has
been peptonized, but still absorption of the native proteid is possible
without the agency of proteolytic enzymes. When, however, large amounts
of egg-albumin are introduced into the intestine, albuminuria may
result, as you very well know.

[199] Ueber die Aufsaugung im Dick und Dünndarm, Zeitschr. f. Biol.,
Band 5, p. 562.

[200] Ueber den Nährwerth der Eierklystiere, Arch. f. klin. Med., Band
47, p. 495.

Moreover, it is well known that the proteids of muscle-tissue, in the
form of syntonin, may be absorbed from the large intestine without
undergoing further hydration. When introduced into the rectum of a
hungry dog, the excretion of urea may be at once increased and the
animal brought into a condition of nitrogenous equilibrium; absorption
taking place from a portion of the large intestine, where proteolysis
is never known to occur.[201]

[201] Eichhorst: Ueber die Resorption der Albuminate im Dickdarm,
Pflüger’s Archiv f. Physiol., Band 4, p. 570.

Again, Neumeister[202] has shown that the direct introduction of
syntonin, alkali-albuminate, crystalline phytovitellin, as well as
pure serum-albumin, into the blood of the jugular vein is not attended
with the appearance of albumin in the urine. On the contrary, the
proteid matter so introduced appears to be assimilated and utilized
for the needs of the organism. Evidently, then, these substances are
not to be considered as foreign bodies, for if so the kidneys would
undoubtedly make some effort to remove them from the circulation. It is
to be noted, however, that all native proteids are not assimilated in
this manner, as casein,[203] gelatin,[204] and especially egg-albumin.
Thus, J. C. Lehman,[205] working under Kühne’s direction, observed
that the injection of a carefully filtered solution of egg-albumin into
the veins of a dog was always accompanied by albuminuria, while similar
injections of Lieberkühn’s sodium albuminate, or of syntonin from
frog’s muscle, failed to show any such result.

[202] Zur Physiologie der Eiweissresorption und zur Lehre von den
Peptonen, Zeitschr. f. Biol., Band 27, p. 309.

[203] Neumeister: Sitzungsber. der Physik. med. Gesellsch. zu Würzburg,
1889, p. 73.

[204] F. Klug: Pflüger’s Archiv f. Physiol., Band 48, p. 122.

[205] Virchow’s Archiv, Band 30, p. 593.

While these observations tend to show that some native proteids may
be absorbed from the alimentary tract without previously undergoing
proteolysis, it is not to be understood that any considerable quantity
is so absorbed under normal circumstances. Doubtless, when small
amounts of proteid food are taken, its denaturalization by the primary
action of the gastric or pancreatic juice, viz., its conversion into
syntonin or alkali-albuminate, may be sufficient to insure its partial
absorption, but digestive proteolysis is unquestionably a necessary
preliminary to any general absorption, and there can be no manner of
doubt that the greater portion of the proteid food is absorbed as
proteoses and peptone. Peptones, as we have seen, are possessed of
a higher endosmotic equivalent than the proteoses, but we need to
keep continually in mind the possibility that the selective power of
the epithelial cells of the intestinal mucosa may lead to as rapid
transference of the proteoses as of the more diffusible peptones. It
is not to be understood by this, however, that diffusibility is of no
consequence in determining the rate of absorption. Surely, everything
else being equal, the more diffusible the substance the more rapid will
be its passage from the intestine into the blood-current. The more
the process of absorption is studied, however, the more clearly do we
see its dependence upon the functional power of the living epithelial
cells, a fact which plainly emphasizes the physiological nature of the
process.

Further, as already stated, absorption of proteid food-stuffs, or their
products, from the alimentary tract, is, under ordinary circumstances
at least, limited to the intestine; from the stomach there is
comparatively little absorbed, and if necessary we might advance this
fact as an important argument against the theory of general absorption
of proteids in the form of acid-albumin. Even such indifferent fluids
as water, or physiological salt solution, are absorbed with extreme
slowness from the stomach;[206] this organ showing very little ability
to take up water even when the blood-vessels are dilated, as after the
ingestion of food.

[206] J. S. Edkins: The Absorption of Water in the Alimentary Canal.
Journal of Physiol., vol. 13, p. 445.

This brings us to a very important point in connection with the
utilization by the system of the ordinary products of proteolysis.
The latter, as we have seen, are mainly proteoses and peptones, and
yet all the evidence points clearly to the fact that these substances
are never present, at least in any quantity, in the blood or lymph,
even when digestive proteolysis is at its height. Further, the very
nature of the proteoses and peptones, their marked physiological action
when they are introduced directly into the circulating blood, their
rapid excretion, either as proteoses or peptones, by the kidneys when
so introduced,[207] all indicate that they are foreign substances,
totally out of their natural environment when introduced into the
blood-current. And yet we very well know that proteoses especially are
possessed of high nutritive qualities; they are abundantly able to
support animal life. Thus, Politzer[208] found by feeding experiments
with heteroalbumose, dysalbumose, and protoalbumose, that these bodies
taken into the stomach have the same nutritive value as meat. Various
feeding experiments with proteoses from different sources, carried out
in my laboratory on young dogs, have shown conclusively that for short
periods of time, at least, these hydrolytic cleavage products are fully
as capable of sustaining the nitrogenous equilibrium of the body as the
proteids from which they are derived. In fact, the results obtained
favor the view that the proteoses, weight for weight, possess a higher
nutritive value than fresh beef.[209] It may be questionable, however,
whether such a result would follow in experiments conducted over longer
periods of time, but of this we may be certain, that the proteoses
formed in the alimentary tract can be absorbed and utilized by the
system without their exerting any toxic action whatever.

[207] Franz Hofmeister: Ueber das Schicksal des Peptons im Blute,
Zeitschr. f. physiol. Chem., Band 5, p. 125.

[208] Ueber den Nährwerth einiger Verdauungsproducte des Eiweisses,
Pflüger’s Archiv f. Physiol., Band 37, p. 301.

[209] Compare Hildebrandt, Zur Frage nach dem Nährwerth der Albumosen,
Zeitschr. f. physiol. Chem., Band 18, p. 120.

Consequently, we are forced to the conclusion that these primary
products of proteolysis, so important in the nutrition of the animal
body, must undergo some change during the process of absorption, by
which they are converted into new bodies, less toxic in their nature,
and better adapted for the direct nutritional needs of the organism.
The same statement applies likewise to peptones.

The fact that peptones are not discoverable in the blood and lymph,
even during or after active digestion, was practically ascertained
years ago by such well-known workers as Maly, Adamkiewicz, and others.
The natural supposition following this observation was that the
products of proteolysis underwent some change in the hepatic cells; but
this view was soon shown to be untenable by examination of the portal
blood, which was found to be as free from peptone as the blood of the
hepatic vein. Neumeister,[210] using the more modern methods of work
and with the wider knowledge gained during these latter years, has
shown conclusively that proteoses and peptones are never present in
the blood, even when these substances are contained in the intestine
in fairly large amounts. I can corroborate these statements from the
results of my own experiments in this direction. Thus, I have taken a
dog in full digestion, fed with an abundance of meat, and collecting
the blood from the carotid artery have made a careful examination
for peptone, by the following method: The blood was allowed to flow
directly into a dilute solution of ammonium sulphate, sufficiently
strong to prevent coagulation, and then shaken with ether to rupture
the red blood-corpuscles. The solution, freed from ether, was next
saturated with crystals of ammonium sulphate, by which the proteid
matter was completely precipitated. The clear filtrate was then
concentrated somewhat, the excess of the ammonium salt removed by
filtration, and the filtrate carefully tested for peptone by addition
of a large volume of a saturated solution of potassium hydroxide and
a few drops of a dilute solution of cupric sulphate. The test was
wholly negative, although the intestine of the animal showed the
presence of both peptone and proteoses. This result, as I have said,
is simply confirmatory of work done by others in this direction,
notably Neumeister, and illustrates the statement that peptones are
not to be found in the circulating blood, even after a full proteid
diet. In this connection it is to be remembered that we have abundant
proof of the rapid disappearance of both proteoses and peptones[211]
from the intestine, either by absorption or otherwise. They certainly
disappear, and, as we have seen, are not to be found in the blood.
Further, Neumeister has confirmed the original observation of
Schmidt-Mulheim,[212] that both chyle and lymph are practically free
from proteoses and peptone, thus again forcing us to the conclusion
that the primary products of proteolysis must undergo change prior to
their passage into the blood or lymph.

[210] Ueber die Einführhrung der Albumosen und Peptone in den
Organismus, Zeitschr. f. Biol., Band 24, p. 277.

[211] Rohmann: Ueber Secretion und Resorption im Dünndarm, Pflüger’s
Archiv f. Physiol., Band 41, p. 440.

[212] Du Bois-Reymond’s Archiv f. Physiol., p. 33, 1880.

Many observations lend favor to the view that a transformation of
some kind takes place in the intestine itself, not indeed in the
lumen of the tube, but somewhere in the walls, through which the
peptones must pass before reaching the blood. Thus, peptones placed in
contact with pieces of the isolated, though still living, intestine,
after a time completely disappear from view,[213] so completely that
no reaction can be obtained even by the most delicate of tests. In
support of this statement I may cite the results of several of my
own experiments which certainly furnish evidence that true peptones
undergo profound alteration by simple contact with the living mucous
membrane of the small intestine. The method employed was similar to
that made use of some years ago in a study of the influence of peptone
on the post-mortem formation of sugar in the liver.[214] A large,
well-nourished rabbit was killed by severing the carotid artery and the
blood collected and defibrinated. Of this, 50 c. c. were mixed with an
equal volume of 0.5 per cent. salt solution containing 1.25 grammes of
pure amphopeptone, prepared from egg-albumin, the mixture obviously
containing 1.25 per cent. of peptone. The fluid was transferred to a
large, roomy flask, provided with a stopper having two holes, in one
of which was fitted a long glass tube reaching below the fluid. The
flask, with its contents, was then placed in a suitable water-bath at a
temperature of 40° C.

[213] Salvioli: Eine neue Methode für die Untersuchung der Functionen
des Dünndarms, Du Bois-Reymond’s Archiv f. Physiol., 1880. Supplement
Band, p. 112. Neumeister: Zur Physiologie der Eiweissresorption und zur
Lehre von den Peptonen, Zeitschr. f. Biol., Band 27, p. 324.

[214] Chittenden and Lambert: Studies in Physiol. Chem., Yale Univer.,
vol. i., p. 171.

The small intestine of the rabbit was carefully separated from the
mesentery and from the pancreatic gland, and the upper portion cut
open and quickly washed free from any contained matter or adherent
secretions, by repeated immersion in 0.5 per cent. salt solution warmed
at 40° C. This was repeated until the tissue was quite free from all
impurities, after which it was cut into small pieces and immersed for
a moment in a 0.5 per cent. solution of sodium chloride containing
1.25 per cent. of peptone. The tissue was then carefully collected on
coarse muslin, allowed to drain, and then quickly transferred to the
flask containing the warm blood and peptone. This mixture was kept at
40° C. for two hours, a slow current of air being bubbled through the
fluid during the entire period. At the expiration of this time the
fluid was separated from the pieces of tissue by nitration through
muslin, and then saturated with ammonium sulphate after the usual
method for the separation of albumoses, etc. On now testing a portion
of the clear filtrate for peptone by the biuret test, not a trace of
a reaction could be obtained. The entire amount of proteid matter
present was precipitated by the ammonium salt, thus showing that the
peptone originally added had been completely transformed into something
precipitable by saturation of the fluid with ammonium sulphate. That
this transformation of the peptone was accomplished mainly through the
action of the intestine, was shown by a parallel experiment, in which
all of the above conditions were duplicated, omitting only the pieces
of intestine. Here, however, on testing the filtrate from the ammonium
sulphate-precipitate, a strong biuret reaction was obtained, thus
proving the presence of at least some unaltered peptone.

This experiment is almost a counterpart of one reported by Neumeister,
and like his, testifies to the probability that the peptones formed in
the alimentary tract, as a result of proteolysis, undergo retrogression
through the agency of the epithelial cells of the intestinal walls
during their absorption. I have tried similar experiments with
deuteroproteose, notably with deuterocaseose, and have obtained
corresponding results. The same method may be employed as that already
outlined, although of course the deuterocaseose is in great part
precipitated by saturation with ammonium sulphate. Still, this form
of deuteroproteose, β deuterocaseose, as I have elsewhere noted, is
very slowly precipitated by the ammonium salt. Consequently, it is an
easy matter to demonstrate that this proteose, on treatment with the
intestinal mucosa in the presence of blood at the body-temperature,
is transformed into something completely and readily precipitable by
ammonium sulphate; the filtrate from the latter failing to show any
biuret reaction, although the corresponding control experiment without
the intestine gives a bright violet color with cupric sulphate and
potassium hydroxide.

Hence, we are certainly justified in saying that both peptones and
proteoses undergo some retrogression when in contact with the walls of
the intestine. Moreover, there is some evidence that the proteoses,
before undergoing such a transformation, are first converted into
peptone by the action of the intestinal walls, a statement which will
apparently apply to both the primary and secondary proteoses. This
primary action of the intestinal walls is not considered as due to any
adherent trypsin, or to possible traces of succus entericus, but rather
as a part of the action of the living epithelial cells, or perhaps as
connected with the possible presence of lower organisms not removable
from the intestinal wall by ordinary washing.

The transformation of peptones by the substance of the intestine
is apparently common to the intestinal tract of many animals, and
perhaps to all, and indeed can also be accomplished by the liver.[215]
This latter fact is of some importance, since it adds weight to the
supposition that this peculiar action of the intestine cannot be due
to the possible presence of trypsin; a view which is strengthened by
the fact that a glycerin-extract of the intestine has no action on
amphopeptone. Certainly, the latter shows no diminution in the strength
of the biuret reaction after long contact at body temperature with
such an extract. Further, it has been shown that antipeptone, which
is not affected by the pancreatic enzyme, suffers the same change
as amphopeptone by contact with the intestine. Far more probable is
it that retrogression or transformation of peptone by the substance
of the intestine, is due to the vital activity of some or all of
the epithelial cells of the intestinal mucosa; a characteristic
possibly shared by some or all of the hepatic cells of the liver. The
kidney-cells certainly do not possess this power, but we can see a
special fitness in the liver-cells being endowed with the ability to
quickly break down, or transform, any peptone or proteose that might by
chance escape unaltered from the intestinal tract. Shore,[216] however,
inclines to the view that the hepatic cells do not possess this power
to any great extent, in opposition to the older views of Plòsz and
Gyergai,[217] as well as of Seegen[218] and of Neumeister.

[215] Neumeister: Zeitschr. f. Biol., Band 27, p. 332.

[216] On the Fate of Peptone in the Lymphatic System, Journal of
Physiol., vol. xi., p. 528.

[217] Ueber Peptone und Ernährung mit denselben, Pflüger’s Archiv f.
Physiol., Band 10, p. 536.

[218] Zur Umwandlung des Peptons durch die Leber, _Ibid._, Band 37,
p. 325.

With reference to the action of the stomach-mucosa on proteoses, it
has been shown[219] that when relatively large amounts (5 grammes)
are introduced into the stomach of a rabbit, the pylorus being
ligatured, both proteoses and peptones may appear in the urine, thus
indicating that while they may be absorbed to some extent under the
above conditions, the proteoses are not readily transformed into
native proteids without exposure to the intestine. Smaller amounts
(2 grammes), however, may, under the above conditions, be completely
transformed; at least Hildebrandt claims this to be the case, mainly on
the ground that after the introduction of albumoses into the stomach,
the pylorus being ligatured, no trace of them can be found in the
urine. The same observer also claims that blood-serum, in the case of
dogs, is able to transform albumoses into ordinary serum-globulin.
Certainly, after intra-venous injection, proteoses disappear from the
blood, but, as we shall see later on, a certain amount, at least, may
be transferred to the lymph. It is also claimed that when albumoses
are injected subcutaneously, neither albumoses nor peptones are to be
detected in the urine. This, however, seems hardly probable in the
light of what has been said, and especially in view of the fact that
Neumeister’s experiments tend to show that even 0.1 gramme of albumoses
introduced subcutaneously may give rise to temporary albuminuria.

[219] Hildebrandt: Zur Frage nach dem Nährwerth der Albumosen,
Zeitschr. f. physiol. Chem., Band 18, p. 180.

Assuming for the moment that the chief products of proteolysis,
_i. e._, the proteoses and peptones, are, during the act of absorption,
transformed through the vital processes of the epithelial cells of the
intestine into serum-albumin, or globulin, and absorbed as such into
the blood, we may well consider whether such transformation, _i. e._, a
retrogression into a native proteid again, is inconsistent, or out of
harmony, with the general character of the changes known to occur in
the body. In attempting to answer this question we need not look far
to find a perfectly analogous case. Thus, in the digestion of starchy
foods by the amylolytic ferments of both the saliva and the pancreatic
juice, the carbohydrate material undergoes hydration with formation
of dextrins and maltose, the latter, at least, being quickly absorbed
into the circulating blood. But large quantities of sugar in the blood
are certainly inimical to the well-being of the organism, and we find
in the liver a tendency for the sugar to undergo a transformation,
_i. e._, a retrogression into glycogen, either through simple
dehydration or otherwise. Further, with reference to the possible
conversion of proteoses into peptone by the substance of the intestine,
we have a perfectly analogous case in the behavior of the intestinal
mucous membrane toward maltose, the final product of amylolytic action.
Thus, according to the recent work of M. C. Tebb,[220] the mucous
membrane of the intestine has the power of transforming maltose into
dextrose; simple warming at 40° C. of a solution of maltose in 0.5 per
cent. sodium carbonate with a few grammes of the dried mucous membrane
from the intestine, being sufficient to insure a marked conversion of
maltose into the higher-reducing sugar, dextrose. This observation,
I can confirm from experiments just completed in my own laboratory.
This action is presumably due to a ferment, which, according to Tebb,
is widely distributed throughout the body, being present not only
in the intestine, but also in the liver, kidney, spleen, striated
muscle-tissue, and, indeed, in the blood-serum; so that it would appear
that nearly all the tissues of the body are endowed with the power of
transforming maltose into dextrose. These statements being correct, it
would seem that, while the amylolytic ferments of the several digestive
juices transform, by hydrolytic action, starchy foods into maltose,
the latter is exposed during its passage through the intestinal wall,
as well as in the blood itself, to another ferment which carries the
hydration still further, with formation of dextrose; and yet the latter
product is destined, in part at least, to undergo retrogression into a
starch-like body, _i. e._, glycogen, before it is completely utilized
by the system. Thus, the analogy between these carbohydrate bodies and
the products of proteolysis is complete, and we may well accept the
statements already made regarding the ultimate fate of the proteoses
and peptones formed during proteolysis, as in no way inconsistent with
the general tenor of events going on in the body.

[220] On the Transformation of Maltose to Dextrose, Journal of
Physiol., vol. xv, p. 421.

While we are inclined to believe that the chemical changes attending
the absorption of proteoses and peptones occur mainly in the epithelial
cells of the intestinal mucosa, and that there is a direct transference
of the alteration-products to the blood, there are still other views
that cannot be wholly ignored. Thus, the view originally advanced by
Hofmeister,[221] in which special stress is laid upon the functional
activity of the leucocytes of the adenoid tissue surrounding the
intestine, demands some consideration. The theory supposes that these
cells not only have the power of taking up peptones, but also of
assimilating and transforming them into the cell-protoplasm. This view
being correct, it is plain that the so-absorbed proteid must pass into
the circulating blood through the thoracic duct, and Hofmeister further
considers it probable that the lymph-cells of the mesenteric glands can
transform any absorbed peptone that may escape the leucocytes of the
adenoid tissue.

[221] Zeitschr. f. physiol. Chem., Band 4.

In apparent harmony with this view is the fact that the leucocytes in
the adenoid tissue of the intestine are greatly increased in number
during digestion.[222] Furthermore, it is a well authenticated fact
that the proteoses or peptones found in pus are contained in the
pus-cells themselves, and not in the fluid in which the corpuscles
float.[223] In support of the first statement, Pohl,[224] in his recent
study of the absorption and assimilation of food-stuffs, has emphasized
the marked increase in the number of white blood-corpuscles in the
circulating blood after the ingestion of proteid foods, especially
such as meat, Witte’s peptone, and gelatin-peptone. It is to be noted
further that the increase is most marked at about the third hour after
the taking of food, viz., at a time when digestive proteolysis would
naturally be at its height. Moreover, the maximal increase, according
to Pohl’s data, is astounding, amounting as it does in many cases to
a hundred per cent. Thus, in one instance, in the case of a dog, the
number of white blood-corpuscles per cubic millimetre of blood was
8,689; yet two hours after the feeding of 100 grammes of meat the
number increased to 17,296 per cubic millimetre, followed six hours
after by a return to the original figure.

[222] Arch. f. Exp. Pathol. u. Pharm., Band 20 and Band 22.

[223] Zeitschr. f. physiol. Chem., Band 4, p. 268.

[224] Arch. f. Exp. Pathol. u. Pharm., Band 25, p. 31.

This indicates the general tenor of Pohl’s results, which have been
taken, by some physiologists at least, as confirmatory of Hofmeister’s
views; the interpretation naturally being that digestive proteolysis
in the alimentary tract is accompanied by a rapid production of new
leucocytes in the lymph-spaces surrounding the intestine, and followed
by a rapid transference of the corpuscles from their point of origin
to the circulating blood, from which they gradually disappear as
their material is made use of in the different parts of the body. In
harmony with this view, Pohl finds that there is a much larger number
of leucocytes in the blood and lymph flowing from the intestine of
an animal in full digestion, than in the arterial blood coming to
the intestinal tract. Further, when due consideration is given to
all the circumstances attending the circulation of the blood through
the abdominal organs, in connection with the great increase in the
number of leucocytes during digestive proteolysis, it seems not
unreasonable to suppose that some proteid matter might be transferred
from the intestine to the blood during the digestive period of six
or eight hours. Moreover, if Pohl’s views are correct, we see that
a portion, at least, of the proteid food-product may be transformed
into organized material in the body of the lymph-cell prior to its
passage into the blood, thus harmonizing with the statement already
made regarding the utter lack of proteoses and peptones in the blood
and lymph. This obviously means an upbuilding of the ordinary products
of digestive proteolysis into the living protoplasm of the leucocytes
in the intestinal walls, implying, however, that the transformation
is accomplished solely by the leucocytes themselves, and not by the
epithelial cells of the intestine.

I have given this brief summary of Pohl’s work because it is so closely
in harmony with the original views of Hofmeister, and because it
offers an easy explanation of one possible way in which some of the
products of digestion might perhaps pass from the intestine into the
blood. I am inclined to believe, however, that the so-called digestive
leucocytosis, which unquestionably does exist, is not a direct result
of digestive proteolysis in general, but rather an indirect result,
coming from the stimulating action of the nuclein, contained especially
in animal cells. Thus, it is a significant fact, as Pohl himself
reports, that wheat-bread, with its fairly large amount of proteid
matter, and which is fully capable of nourishing the animal body, fails
to exert any influence on the number of leucocytes in the blood. Yet we
know that the gluten and other proteids of wheat-flour are converted
by digestive proteolysis into proteoses and peptones, with the same
general properties as like products of animal origin.

In this connection we may note the experiments of Horbaczewski,[225]
which show that nuclein administered to a healthy man will give rise
to a very marked increase in the number of leucocytes in the blood.
Thus, a few grammes of nuclein may produce as striking a condition
of leucocytosis as a large amount of proteid food, due no doubt to
proliferation of the lymphoid elements of all the lymphatic tissues
of the body. Horbaczewski has reported that the mere injection of
0.25 gramme of nuclein, in the case of rabbits, will cause marked
enlargement of the spleen, with striking karyokinetic changes.
Hence, it may be assumed that whenever nuclein is set free in the
body, leucocytosis may result, provided the nuclein passes into the
circulation and is not decomposed immediately after its liberation.

[225] Monatshefte f. Chemie, Band 12, p. 246.

These facts, it appears to me, offer a more consistent line of
explanation of digestive leucocytosis than that advanced by Pohl.
All animal foods, especially meat of various kinds and milk, contain
considerable nuclein or nucleo-albumins, which, by the action of the
gastric juice, are liberated and partially digested, but the nuclein
is certainly not dissolved. Nucleins, however, are soluble in weak
alkaline fluids, and when exposed to the action of the alkaline
pancreatic juice in the intestine, are in great part dissolved. Thus,
Popoff[226] has reported that different varieties of nuclein behave
somewhat differently in the intestine, according to their origin. In
young and tender tissues, solution of the contained nuclein through the
alkaline fluids of the intestinal canal is fairly complete, while the
older products are somewhat more resistant both to the pancreatic juice
and to the putrefactive processes common to the intestine. However,
experiments show that the greater portion of the nuclein of ordinary
proteid foods is dissolved in the intestine, and absorbed as such in
a practically unaltered form. Consequently, passing into the adenoid
tissue surrounding the intestine, it has a marked stimulating action
on the lymphoid elements, accompanied by a noticeable increase in the
number of leucocytes, which are perhaps produced at the expense of a
portion of the proteoses and peptones formed during proteolysis.

[226] Ueber die Einwirkung von Eiweissverdauenden Fermenten auf die
Nucleinstoffe, Zeitschr. f. physiol. Chem., Band 18, p. 533.

Thus, my interpretation of these results would lead me simply to the
admission that, possibly, a portion of the products of proteolysis
might pass from the intestine into the blood-current indirectly,
through the bodies of the leucocytes formed in the adenoid tissue
of the intestine. But even admitting this, we lack positive proof
of any direct transformation of proteoses and peptones into the
organized material of the white blood-corpuscles, for it may be that
the above products are first transformed through other agencies into
serum-albumin, or other like proteids. There are, indeed, many facts
which are plainly opposed to any marked absorption and transformation
of peptones by the leucocytes of the intestinal mucous membrane. Thus,
Heidenhain[227] has severely criticised the theory on the ground that
there is very little increase in the flow of lymph from the thoracic
duct during absorption, and further that the small percentage of
proteid matter in the chyle (about 2.0 per cent.) cannot account for
the large amount of proteid absorbed. Further, the objection is made
that the leucocytes present in the intestinal mucosa, though numerous,
are wholly inadequate to assimilate any large proportion of the
ingested proteid food.

[227] Beiträge zur Histologie und Physiologie des Dünndarms, Pflüger’s
Archiv f. Physiol, Band 43, Supplement Heft.

Still greater stress, however, may be laid upon the fact that
experimental evidence points to the conclusion that lymph-cells
cannot assimilate either peptones or proteoses. Thus, quite recently,
Shore[228] has studied the results following the introduction of a
mixture of such products into the lymphatic system by secretion,
by absorption, and by direct injection into a lymphatic vessel.
Preliminary experiments on dogs showed that when peptone is introduced
into the bile-duct it gradually appears in the lymph of the thoracic
duct, consequently this method can be made use of as a means of
ascertaining the fate of peptone so absorbed into the lymphatic system.
The results obtained, using the ammonium sulphate method for the
isolation of the peptone, showed that when peptone is injected into
the bile-duct with sufficient force to overcome the low pressure under
which bile is secreted, there is an increase in the rate of flow of
lymph from the thoracic duct. Further, while peptone is somewhat slow
in appearing in the lymph it eventually makes its appearance there, in
from sixty to one hundred and forty minutes after its injection into
the bile-duct. A certain amount of peptone naturally passes into the
blood, but is then rapidly excreted through the urine. When, however,
the renal vessels are ligatured, peptone still rapidly disappears from
the blood, but then passes into the lymph, and under such circumstances
can be detected in the lymph as early as thirty-eight minutes after its
injection into the bile-duct. These results, therefore, do not accord
with the view that peptones suffer marked transformation by contact
with lymph-cells, for when only three-fourths of a gramme of peptone
is introduced into the bile-duct, unaltered peptone can be detected
in the lymph of the thoracic duct seventy to ninety minutes after its
injection.

[228] On the Fate of Peptone in the Lymphatic System, Journal of
Physiol., vol. xi., p. 528.

With reference to the fate of peptone when it passes by secretion into
the lymphatic system, it will be remembered that Heidenhain[229] has
shown that the injection of peptone into the blood may be followed by
a large increase in the rate of flow of lymph. Further, the amount of
solids in the lymph, especially of proteids, is considerably increased.
From these and other facts, Heidenhain is led to the view that the
formation of lymph is a true secretion from the blood-vessels. Shore
finds that when small amounts of peptone are slowly injected into the
blood, there is generally only a slight acceleration in the flow of
lymph, but the clotting power of the lymph is affected in a remarkable
manner. Thus, about twenty minutes after the commencement of the
injection the lymph loses entirely its power of coagulating. This
continues for about twenty minutes, and then, in spite of the fact that
the injection is being continued, the lymph rapidly regains its power
of clotting, and finally coagulates quicker and firmer than before.
This peculiar action of peptone on the clotting power of lymph may
frequently be observed, even when the amount of peptone present is too
small to be detected with certainty by chemical methods. Thus, when
peptone in small quantity is injected very slowly into the blood, the
greater part of it escapes through the urine, but a small fraction,
sometimes too small to actually detect, passes into the lymph and shows
its presence by its peculiar influence on the clotting of the fluid.

[229] Versuche und Fragen zur Lehre von der Lymphbildung, Pflüger’s
Archiv f. Physiol., Band 49, p. 252.

When, on the other hand, peptone is injected rapidly into the blood,
0.3 to 0.6 gramme per kilo. during two to ten minutes, it may disappear
completely from the blood in five to ten minutes after the end of the
injection. In such a case, the fall of blood-pressure induced leads to
more or less arrest of the renal secretion, peptone appearing in large
amount in the lymph; but there is no indication of any alteration of
the peptone by the lymph, or its contained leucocytes. Thus, when there
is no chance for the peptone to escape from the body, as on ligation
of the renal vessels, the peptone injected into the blood is rapidly
thrown into the lymph, and from the lymph in the tissues of the body it
is gradually carried to the thoracic duct, and then again passes into
the blood; all of which shows that there is little or no transformation
of the peptone by the leucocytes of the lymphatic system.

Further, by direct injection of peptone into a lymphatic vessel,
Shore has shown that even so small an amount as 0.049 gramme is not
assimilated or transformed by the lymph in half an hour. Consequently,
we seem to have strong evidence that peptones are not prone to direct
alteration of any kind by the leucocytes of the lymphatic system.
Further, it would appear that the lymphoid cells of the spleen are
equally unable to assimilate small amounts of peptone injected into
the splenic artery. Leucocytes, then, can play no direct part in the
absorption of the products of proteolysis from the intestine; the lymph
is normally free from both proteoses and peptones, and the leucocytes
plainly have no power to transform these bodies into other forms. They
can only utilize the proteid material elaborated from the products of
proteolysis by other agencies.

Plainly, proteoses and peptones in the blood and lymph are foreign
substances. When present in the circulation they give rise, as we
have seen, to an increased flow of lymph and to a change in the
coagulability of the blood. Further, not only is the flow of lymph
augmented but there is likewise an increase in the amount of solid
matter, while a corresponding decrease is noticed in the solid matter
of the blood-plasma. This fact obviously gives support to the view
that the increased formation of lymph after the injection of peptone
is due to an active process of secretion by the endothelium cells of
the capillary walls.[230] Further, as we have seen, peptone disappears
from the blood more or less rapidly after its injection, so that it
is quite possible that the loss or alteration of the coagulability
of the blood may not be due to the peptone itself, but rather to an
altered condition of the blood induced by the peptone. Moreover, this
altered condition of the blood may be the real cause of the increased
transudation, or secretion of lymph so conspicuous after the injection
of peptone. Starling, however, by carefully conducted experiments
on dogs finds, in conformity with Heidenhain’s views, that peptone
injected into the blood exercises a direct excitatory effect on the
endothelial cells, causing thereby an increased flow of lymph; the
increased flow being in no way caused by the change in the blood that
is simultaneously produced. Further, it would appear, according to
Starling’s views, that the change in the coagulability of the blood is
not due to the effect of the peptone on the endothelial cells of the
blood-vessels, or at least on their lymph-producing functions. Thus,
the injection of peptone may result in an action on the endothelial
cells of the blood-vessels, thereby increasing the flow of lymph, or
on the blood itself with a destruction or diminution of the clotting
power of the blood; the two results being more or less independent.
Further, the rapid transferences of peptone from the blood to the
lymph is effected by the selective activity of the endothelial cells
of the vessel-wall, and according to Starling it is probable that a
preponderating part is played by the endothelial cells of the renal
capillaries.

[230] Starling: Contributions to the Physiology of Lymph Secretion,
Journal of Physiol., vol. xiv, p. 131.

In view of all these statements, it is very evident that proteoses and
peptones once outside the limits of the alimentary tract may be passed
about from organ to organ and from secretion to secretion, inducing
changes here and there in their course, but suffering very little
change themselves. The main efforts of the system are directed to the
removal of these unwelcome strangers as speedily as possible, for their
marked physiological action renders them somewhat dangerous visitors.

As normal products of digestive proteolysis, they are never found
beyond the limits of the gastro-intestinal canal, but undergo
retrogression in their passage through the epithelial cells of
the intestinal wall, being presumably converted thereby into
serum-albumin,[231] which can be directly utilized for the nutrition of
the body; a conversion which is plainly dependent upon certain inherent
qualities of the living epithelial cells, and is doubtless of the
nature of a dehydration.

[231] See Kronecker and Popoff: Ueber die Bildung von Serumalbumin im
Darmkanale, Du Bois-Reymond’s Archiv f. Physiol., 1887, p. 345. Also
Nadine Popoff, Zeitschr. f. Biol., Band 25, p. 427.





End of Project Gutenberg's On Digestive Proteolysis, by R. H. Chittenden

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