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.
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.
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.
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.
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.
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.
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.
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.
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]
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.
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.
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.
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.”
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 H2O + O2 = CO(OH)2 + H2O2.
The hydrogen-peroxide thus formed combines with carbonic oxide to form carbonic acid, which in turn is decomposed into the anhydride CO2, with regeneration of water, the latter being available for further action of the same order:
H2O2 + CO = CO(OH)2
2 CO(OH)2 = 2 CO2 + 2 H2O.
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, CO2), 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.
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.
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]
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.
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.
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]
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.*
| Substance. | C | H | N | S | O | P | Ash. | Origin. | Author. | |
| Serum-albumin | 63.05 | 6.85 | 16.04 | 1.77 | 22.29 | .... |  | 0.57— | Serum from horse blood | Hammarsten.[54] |
| Serum-albumin | 52.25 | 6.65 | 15.88 | 2.27 | 22.95 | .... | 1.84 | Pleural exudation | Hammarsten.[54] | |
| Egg-albumin | 52.25 | 6.90 | 15.25 | 1.93 | 23.67 | .... | .... | Non-coagulated | Hammarsten.[54] | |
| Egg-albumin | 52.33 | 6.98 | 15.89 | 1.83 | 22.97 | .... | 1.11 | Non-coagulated | Chittenden and Bolton.[55] | |
| Lacto-albumin | 52.19 | 7.18 | 15.77 | 1.73 | 23.13 | .... | .... | Cow’s milk | Sebelien.[56] | |
| Vegetable-albumin | 52.25 | 6.76 | 16.07 | 1.48 | 23.44 | .... | 0.70 | Corn or maize | Chittenden and Osborne.[57] | |
| Vegetable-albumin | 53.02 | 6.84 | 16.80 | 1.28 | 22.06 | .... | 0.82 | Wheat | Osborne and Voorhees.[58] | |
| Proteose, animal | 52.13 | 6.83 | 16.55 | 1.09 | 23.40 | .... | 0.79 | Hemialbumose, urine | Kühne and Chittenden.[59] | |
| Proteose, vegetable | 60.60 | 6.68 | 16.33 | 1.62 | 24.77 | .... | 2.99 | Corn or maize | Chittenden and Osborne.[57] | |
| Proteose, vegetable | 51.86 | 6.82 | 17.32 | .... | .... | .... | 0.25 | Wheat | Osborne and Voorhees.[58] | |
| Proteose, vegetable | 49.98 | 6.95 | 18.78 | .... | .... | .... | 1.80 | Flax-seed | Osborne.[60] | |
| Proteose, vegetable | 46.52 | 6.40 | 18.25 | .... | .... | .... | 2.20 | Cocoanut meat | Chittenden and Setchell.[61] | |
| Vitellin, spheroidal | 51.71 | 6.84 | 18.12 | 0.85 | 22.48 | .... | 1.20 | Corn or maize | Chittenden and Osborne.[57] | |
| Vitellin, crystalline | 51.60 | 6.97 | 18.80 | 1.01 | 21.62 | .... | 0.30 | Squash-seed | Chittenden and Hartwell.[62] | |
| Vitellin, amorphous | 51.81 | 6.94 | 18.71 | 1.01 | 21.53 | .... | 0 | Squash-seed | Chittenden and Hartwell.[61] | |
| Vitellin, crystalline | 51.48 | 6.94 | 18.60 | 0.81 | 22.17 | .... | 0.54 | Flax-seed | Osborne.[60] | |
| Vitellin, spheroids | 51.03 | 6.85 | 18.39 | 0.69 | 23.04 | .... | 0.49 | Wheat | Osborne and Voorhees.[58] | |
| Vitellin, crystalline | 51.63 | 6.90 | 18.78 | 0.90 | 21.79 | .... | 0.56 | Hemp-seed | Chittenden and Mendel.[61] | |
| Vitellin, crystalline | 51.31 | 6.97 | 18.75 | 0.76 | 22.21 | .... | 0.03 | Castor bean | Osborne.[63] | |
| Vitellin, crystalline | 52.18 | 6.92 | 18.30 | 1.06 | 21.54 | .... | 0.20 | Brazil nut | Osborne.[63] | |
| Vitellin, semi-crystalline | 51.23 | 6.90 | 18.40 | 1.06 | 22.41 | .... | 0.25 | Cocoanut meat | Chittenden and Setchell.[61] | |
| Myosin, 13 different samples | 52.82 | 7.11 | 16.77 | 1.27 | 21.90 | .... | 1.45 | Muscle-tissue | Chittenden and Cummins.[64] | |
| Myosin, vegetable | 52.68 | 7.02 | 16.78 | 1.30 | 22.22 | .... | 0.63 | Corn or maize | Chittenden and Osborne.[57] | |
| Myosin, vegetable, crystalline | 52.18 | 7.05 | 17.90 | 0.53 | 22.34 | .... | 0.10 | Oats | Osborne.[65] | |
| Paraglobulin | 52.71 | 7.01 | 15.85 | 1.11 | 23.24 | .... | 0.30 | Blood of horse | Hammarsten.[66] | |
| Fibrinogen | 52.93 | 6.90 | 16.66 | 1.25 | 22.26 | .... | 1.75 | Blood of horse | Hammarsten.[67] | |
| Zein | 55.23 | 7.26 | 16.13 | 0.60 | 20.78 | .... | 0.43 | Corn or maize | Chittenden and 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.[65] | |
| Glutenin | 52.34 | 6.83 | 17.49 | 1.08 | 22.25 | .... | .... | Wheat | Osborne and Voorhees.[58] | |
| Coagulated proteid | 52.33 | 6.98 | 15.84 | 1.81 | 23.04 | .... | 0.27 | Egg-albumin | Chittenden and Bolton.[55] | |
| Coagulated proteid | 51.58 | 6.88 | 18.80 | 1.09 | 21.65 | .... | 0.25 | Vitellin, hemp-seed | Chittenden and Mendel.[61] | |
| Fibrin | 52.68 | 6.83 | 16.91 | 1.10 | 22.48 | .... | 0.56 | Blood of horse | Hammarsten.[67] | |
| Oxyhæmoglobin | 53.85 | 7.32 | 16.17 | 0.39 | 21.84 | .... | 0.43 Fe. | Blood of dog | Hoppe-Seyler.[68] | |
| Oxyhæmoglobin | 54.71 | 7.38 | 17.43 | 0.48 | 19.60 | .... | 0.39 Fe. | Blood of pig | Hütner.[69] | |
| 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 | Submaxilliary gland | Hammarsten.[71] | |
| Chondromucoid | 47.30 | 6.42 | 12.58 | 2.42 | 31.28 | .... | .... | Cartilage | Mörner.[72] | |
| 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-histon or leuconuclein | 48.41 | 7.21 | 16.85 | 0.70 | 24.41 | 2.42 | .... | Leucocytes | Lilienfeld.[77] | |
| Gelatin | 49.38 | 6.81 | 17.97 | 0.71 | 25.13 | .... | 1.26 | Connective tissue | Chittenden and 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 rabbit’s hair | Kühne and Chittenden.[81] | |
| 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 tissue | Siegfried.[83] | |
* Many of these results represent the average of a large number of individual analyses.
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.
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:
| Egg-albumin. | Antialbumid[87] from egg-albumin. | Serum-albumin. | Antialbumid[87] from 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 |
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 egg-albumin. | Atmidalbumose precipitated by NaCl. | Atmidalbumose precipitated by NaCl + acid. | Deutero- atmidalbumose. | Antialbumid. | |
| 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 | .... |
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]
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.
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.
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.
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.