Part 2

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.