Part 10

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

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

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

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

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

and known as oxyphenyl-amido-propionic acid.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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