Chapter III: ALBUMINOIDS.--A. Soluble in boiling water with formation of (3)

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

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

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

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

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

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

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

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

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

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

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

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

[165] _Ibid._

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

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

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

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

LECTURE III.

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

PROTEOLYSIS BY TRYPSIN.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

COMPOSITION OF ANTIPEPTONES.

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

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

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

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

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

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

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

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

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

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

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

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

and known as oxyphenyl-amido-propionic acid.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Another product of trypsin-proteolysis which has long been recognized, although its real nature has not been known, is tryptophan or proteinochromogen. This body is not only a product of the pancreatic digestion of proteids, but it is also formed whenever native proteids are broken down through any influence whatever, the substance coming presumably from the hemi-moiety of the molecule. It is especially characterized by the bright-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.

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

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

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

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

[198] Lea: loc. cit.

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

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

ABSORPTION OF THE MAIN PRODUCTS OF PROTEOLYSIS.

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

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

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

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

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

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

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

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

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

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

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