Transcriber’s notes:
In this transcription, paired _underscores_ denote _italicised text_. A
single underscore preceding curly brackets indicates that the bracketed
character is subscripted (mostly in chemical formulae, e.g. CO_{2}).
Footnotes have been positioned below the relevant paragraphs.
Punctuation inconsistencies have been corrected silently, except for
those occurring in footnote reference sources, which remain as in the
original.
Inconsistencies of hyphenation, use of italics, and spacing of
abbreviations (such as i.e. and c.c.) have not been changed.
In a large table following page 31, apparent inconsistencies of the
authors of referenced sources are as in the original. Some of the
figures in this table are indistinct.
Passages of German text contain possible typographic errors that have
been left as in the original.
Spelling inconsistencies have not been altered, e.g. albumin/
albumen/albumine/albumid, but the following overt spelling errors
were corrected:
synonomous —> synonymous
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 retards proteolytic action, doubtless in
the same manner that cold checks or retards 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 retards 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.