Part 9

With proteoses, however, different results are obtained, as Neumeister[156] first pointed out. These bodies introduced into the blood undergo more or less of a change prior to their excretion in the urine, the change partaking of the character of a hydrolytic cleavage in which the primary proteoses are transformed into secondary proteoses, while deuteroproteoses are changed into peptones. This is not necessarily to be interpreted as meaning that the full equivalent of the proteose injected appears in the urine, but that the portion which is eliminated through the kidneys tends to undergo a transformation somewhere _en route_, akin to the change produced in pepsin-proteolysis. As to how common or complete this transformation is under the above circumstances, we have no positive knowledge. Such a hydrolytic change certainly occurs in the case of the dog, and the experimental evidence is in favor of the view that the transformation is effected in the kidneys by the pepsin secreted through the urinary tubules, where there is momentarily a formation of free acid. In the rabbit, on the other hand, no such change occurs; the urine from this animal contains practically no pepsin, and consequently the proteoses eliminated through the kidneys are excreted unaltered. As, however, the experiments of Stadelmann[157] and others have shown that the urine of all carnivora, and of man as well, contains a ferment which, on the addition of a suitable amount of hydrochloric acid, will digest fibrin with formation of the ordinary products of pepsin-proteolysis, it is to be presumed that all proteoses passing through the kidneys will undergo at least some change prior to their excretion in the urine.

[156] _Ibid._, p. 284.

[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.