BOUGHT WITH THE INCOMB FROM THE SAGE ENDOWMENT FUND THE GIFT OF 1891 A.a =3° d P-D a 5 caaa a go ansa 03 "-"O n a s 03 HI « « Q m la m V Si -.-.SO d a Bj-d £2a si's P*^ c a " . l: "1- ■ « r ■g - ^ p« tl.r ?g| s 'is ai a ■=■32 i-scfi ■2 ^"^ ^ *j= "CO or* " moo ;'-J :t^«i— o)syoo5»c^ cJ^-3 .i-« -ooocioi-ipir- _ -win ooooo all £ |Ss©poo5S COMPOSITION OF PEOTEIDS. 33 In considering the results tabulated above, it is to be remembered that all of these bodies, with the exception of keratin, neurokeratin, and reticulin, are more or less digestible in either gastric or pancreatic juice, or indeed in both fluids. I will not take time here to point out the obvious genetic relationships and differences in composition shown by the above data, but will immediately call your attention to the fact that there are other and more impor- tant points of difference between many of these proteids which are hidden beneath the surface, and which a simple determination of composition will not bring to light. I refer to the chemical constitution of the bodies, to the way in which the individual atoms are arranged in the molecule, on which hinges more or less the general properties of the bodies and which in part determines their behavior toward the digestive enzymes, as well as toward other hydrolytic agents. These differences in inner structure can only be ascertained by a study of the decomposition products of the proteids, and of the way in which the com- plex molecules break down into simpler. The nature of the fragments resulting from the decomposition of a com- plex proteid molecule, gives at once something of an insight into the character of the molecule. Thus, egg- albumin exposed to the action of boiling dilute sulphuric acid yields, among other fragments, large quantities of leucin and tyrosin, the latter belonging to the aromatic group and containing the phenyl radical. Collagen, or gelatin, on the other hand, by similar treatment fails to yield any tyrosin or related aromatic body, but gives instead glycocoU or amido-acetic acid, in addition to leucin, lysin, and other products common to albumin. Its constitution, therefore, is evidently quite different from that of albumin, but the composition of the body reveals no sign of it. Further, we have physiological evidence of this difference 4 34 DIGESTIVE PE0TE0LT8IS. in constitution in that gelatin, though containing even more nitrogen than albumin, is not able to take the place of the latter in supplying the physiological needs of the body ; its food-value is of quite a different order from that of albumin. But while all of the individual proteids show many points of difference, either in composition, constitution, reactions, or otherwise, they are nearly all alike in their tendency to undergo hydrolytic decomposition under proper conditions; the extent of the hydrolysis and accompanying cleavage being dependent simply upon the vigor or duration of the hydrolytic process. Furthermore, all of the simple proteids, at least, give evidence of the presence of two distinct groups or radicals, which give rise by decomposition or cleavage to two dis- tinct classes of products. These two groups, which we may assume to be characteristic of every typical proteid, Klihne has named the anti- and hemi-group respectively. This conception of the proteid molecule is one of the foundation-stones on which rest some of our present theo- ries regarding the hydrolytic decomposition of proteids, especially by the proteolytic enzymes. Moreover, it is not a mere conception, for it has been tested so many times by experiment that it has seemingly become a fact. The two groups, or their representatives, can be separated, in part, at least, by the action of dilute sulphuric acid (three per cent.) at 100° C. Thus, after a few hours' treatment of coagulated egg-albumin, about fifty per cent, of the proteid passes into solution, while there remains a homogeneous mass, something like silica in appearance, insoluble in dilute acid, but readily soluble in dilute solutions of sodium carbonate. This latter is the representative of the anti- group, originally named by Schiitzenberger ' hemiprotein, ^ Eeoherolies sur I'albumine et les matiferes albuminoides. Bulletin de la SocidW ohlmique de Paris, vols. 33 and 24. DECOMPOSITION OF PEOTEIDS. 35 but now called antialbumid.' It is only slightly digestible in gastric juice, but is readily attacked by alkaline solutions of trypsin, being converted thereby into a soluble peptone known as antipeptone. In the sulphuric acid solution, on the other hand, are found the representatives of the hemi- group; viz., albumoses, originally known as one body, hemialbumose,' together with more or less hemipeptone, leucin, tyrosin, etc. The fact that we have so many representatives of the hemi-group in this decomposition is significant of the readiness with which the so-called hemi-group undergoes change. All of its members are prone to suffer hydration and cleavage, passing through successive stages until leucin, tyrosin, and other simple bodies are reached. These, and other similar crystalline bodies, are likewise the typical end-products of proteolysis by trypsin, and presuma- bly come directly from the breaking-down of hemipep- tone. Antipeptone, on the other hand, is incapable of further change by the proteolytic ferment trypsin. Hence, the hemi-group can be identified by the behavior of the body containing it toward trypsin ; i. e., it will ultimately yield leucin, tyrosin, and other bodies of simple constitu- tion to be spoken of later on. The anti-group, however, will show its presence by a certain degree of resistance to the action of trypsin, antipeptone being the final product of its transformation by this agent ; i. e., leucia, tyrosin, etc., will not result. In this hydrolytic cleavage of pro- teids the anti-group does not always appear as antialbumid. It may make its appearance in the form of some related body, the exact character of the product being dependent * Kiihne : Weitere Mittheilungen uber Verdauungsenayme viiid die Verdauung der Albumine. Verhandl. d. Natiirhist. Med. Ter. zu Heidelberg, Band 1, p. 236. " Kuhne uud Chittenden : Ueber die nSchsten Spaltungsproducte der Biweisskorper: Zeitschr. f. Biol., Band 19, p. 159. 36 DIGESTIVE PEOTEOLT8I8. in great part upon the nature of the hydrolytic apjent, but in every case the characteristics of the anti-group wUl come to the surface when the body is subjected to the action of trypsin. The above-described treatment of a coagulated proteid with water containing sulphuric acid evidently induces profound changes in the proteid molecule. The conditions are certainly such as favor hydration, and in the case of complex molecules, like the proteids, cleavage might nat- urally be expected to follow. Analysis of antialbumid from various sources plainly shows that its formation is accompanied by marked chemical changes. Thus, the following data, showing the composition of antialbumid formed from egg-albumin and serum-albumin by the action of dilute sulphuric acid at 100° C, gives tangible expres- sion to the extent of this change : Egg-albamin. Antlalbnmldi from eBg-albnmln. Semm-albumln. ADtlalbnmldi from serum-albumin. c H N" 52.33 6.98 15.84 53.79 7.08 14.55 58.05 6.85 16.04 54.51 7.37 14.31 In both cases there is a noticeable decrease in nitrogen, and a corresponding increase in the content of carbon. Evidently, then, this cleavage of the albumin-molecule into the anti-group on the one hand, and into bodies of the hemi-group on the other, is accompanied by chemical changes of such magnitude that their imprint is plainly visible upon the resultant products ; changes which certainly are far removed from those common to polymerization. ' Kttlme und Chittenden : Zeitsohr. f. Biol., Band 19, pp. 167 and 178. FOEMATION OF ATMIDALBUM08E8. 37 This proneuess of proteid matter to undergo hydration and subsequent cleavage is further testified to by the readiness with which even such a resistant body as coagu- lated egg-albumin breaks down under the simple influ- ence of superheated water at 130° to 150° C. Many observations are recorded bearing on this tendency of proteid matter, but few observers have carried their experi- ments to a satisfactory conclusion. A recent study of this question in my own laboratory, has given some very inter- esting results.' Thus, coagulated egg-albumin placed in sealed tubes with a little distilled water and exposed to a temperature of 150° C for three to four hours, rapidly dissolves, leaving, however, an appreciable residue. The solution reacts alkaline, there is a separation of sulphur, and in the fluid is to be foimd not albumin, but two dis- tinct albumose-like bodies, together with some true pep- tone, and a small amount of leucin, tyrosin, and presumably other bodies.' The albumose-like bodies are in many ways quite peculiar. In some respects they resemble the albn- moses formed in ordinary digestion ; but in others they show peculiarities which render them quite unique, so that they merit the specific name of atmidalbumoses, as sug- gested by Neumeister. What, however, I wish to call attention to here is the composition of these albumoses. Prepared from coagulated egg-albumin by the simple action of heat and water, they show a deviation from the composition of the mother-proteid, which plainly implies changes of no slight degree. This is clearly apparent from the following table : ' Chittenden and Meara : A Study of the Primary Products Eesulting from the Action of Superheated Water on Coagulated Egg-albumin. Journal of Physiology, vol. 15, p. 501. ' Compare Neumeister's experiments on blood-fibrin. Ueber die nSchste Einwirkung gespannte WasserdSmpfe auf Proteine und uber erne Gmppe eigenthnmlicher Eiweisskorper und Albumosen. Zeitschr. f. Biol., Band 36, p. 57. 38 DIGESTIVE PEOTEOLYSIS. C i H N ' S O i Coagulated egg-albnmlQ. 53.33 6.98 15.84 1.81 23.04 Atmldalbnmose precipitated by KaCl. 55.13 6.93 14.28 1.66 22.00 Atmidalbnmose precipitated by NaCl + acid. 55.04 6.89 14.17 Deutero- atiDidalbamoBe. 51.99 6.60 13.25 0.98 27.18 Antialbmnid. 53.79 7.08 14.55 Here we see that two of these primary albnmoses formed hj the action of superheated water, like the previously described antialbumid, show a loss of nitrogen with a marked increase in the content of carbon. Evidently, they are related to the antialbumid formed by the action of dilute acid. They are, however, soluble in water, and in many ways differ from true antialbumid, but there is evi- dently an inner relationship. The so-called deuteroatmid- albumose shows a still more noticeable falling off in nitrogen and sulphur, while the content of carbon is more closely allied to that of the mother-proteid. The albumose preeipitable by sodium chloride, although different from an albumid, evidently comes from the anti-group and is a cleavage product which in turn may undergo further hydration and splitting by continued treatment. The so-called deutero-body, on the other hand, may well be a representative of the hemi-group.' It is not my purpose here to enter into details connected with the action of superheated water on proteids. Such a course would take us too far from our present subject, but I do wish to emphasize the fact that even the most resist- ant of proteids has an innate tendency to undergo hydra- tion and cleavage, and that even simple heating with water alone, at a temperature slightly above 100° C, is sufficient ' Compare Ernkenberg, Sitztmgsberiohte der Jenaischen GeseUschaft fiir Medicin, etc. 1886. HYDEO LYTIC CLEAVAGE OF PEOTEIDS. 39 to induce at least partial solution of the proteid. Further, this solvent action in the case of water and dilute acids, at least, is certainly associated with marked chemical changes. It is not mere solution, it is not simply the formation of one soluble body, but solution of the proteid is accompa- nied by the appearance of a row of new products, in which the terminal bodies are crystalline substances of simple composition. Further, this conclusion does not rest upon the results obtained from a single proteid, for I have at various times studied also the primary products formed in the cleavage of casein, elastin, zein, and other proteids by the action of hot dilute acid, and in all cases have obtained evidence of the formation of several proteose-like bodies, as well as of true peptones. By the action of more powerful hydrolytic agents, such as boiling hydrochloric acid to which a little stannous chloride has been added to prevent oxidation, the proteid molecule may be completely broken down into simple decomposition products, of which leucin, tyrosin, aspartic acid, glutamic acid, glucoprotein, lysin, and lysatinin are typical examples.' In other words, by this and other methods of treatment, which we cannot take time to con- sider, we can easily break down the albumin-molecule completely into bodies which, as we shall see later on, are typical end-products of trypsin-proteolysis, and which are far removed from the original proteid. But, as we have seen, even the primary bodies formed in the less profound hydrolysis induced by superheated water, do not show the composition of the mother-proteid. Hydration and cleav- age leave their marks upon the products, and thereby we know that solution of the proteid is the result of some- thing more than a mere rearrangement of the atoms in the molecule. ' Hlasiwetz und Habermarm, Ann. Ohem. u. Pharm. , Band 169, p. 150. Also Dreohsel, Du Bois-Eeymond's Archiv f. Physiol., 1891, p. 355. 40 DIGESTIVE PEOTEOLTSIS. Furtlier, we are to remember that boiling dilute acid and superheated water tend to produce a cleavage along specific lines; viz., a cleavage into the anti- and hemi- groups of the molecule, and as representatives of these groups we may, in the hydration of every native proteid, look for two distinct rows of closely related substances. In digestive proteolysis it will be our purpose to show that cleavage of much the same order occurs, not neces- sarily resulting, however, in the formation of identically the same products, but certainly accompanied with the pro- duction of bodies belonging to the hemi- and anti-groups, although they may be less sharply separated from each other than in the cleavage with dilute sulphuric acid. The body originally described as hemialbumose, and identified as a product of every gastric digestion, is now known to be a mixture of closely related substances ordi- narily spoken of as albumoses,' or generically as proteoses. These are primary products in the digestion of every form of proteid matter, intermediate between the mother-proteid and the peptone which results from the further action of the proteolytic enzymes. Associated with the hemialbumoses are corresponding antialbumoses, coming from the anti- half of the proteid molecule, and differing from their neighbors, the hemi-bodies, mainly in their behavior toward the ferment trypsin. Thus, we have the counter- part of the many bodies described by Meissner, although now arranged systematically and on the basis of structural and other differences not thought of in his day. By the initial action of pepsin-acid, proteids are first transformed into acid-albumin or syntonin, then, by the further action of the ferment, this body is changed into the primary proteoses, proto and heteroproteose, of each of which there must be two varieties, a hemi and an anti. ' Kiihne und Chittenden : TJebei- Albumosen, Zeitsolir. f. Biol., Band 20, p. 11. PEIMAET PEODUCTS OF DIGESTION. 41 These may then undergo further transformation into what is known as a secondary proteose, viz., deuteroproteose, of which there must likewise be two varieties, corresponding to the hemi- and anti-groups respectively. By continued proteolytic action there results as the final product of gastric digestion peptones ; approximately, an equal mix- ture of so-called hemipeptone and antipeptone, generally known as amphopeptone. Such a peptone exposed to the proteolytic action of trypsin should obviously break down in part into simple crystalline bodies, leaving a residue of true antipeptone. In truth, this is exactly what does hap- pen when the peptone resulting from gastric digestion is warmed with an alkaline solution of trypsin. The so-called hemipeptone quickly responds to the action of the pancre- atic ferment, and is converted into other products, while the so-called antipeptone resists its action completely, thus giving results in harmony with our general conception of the proteid molecule. Albumin Molecule. (Hemi-groups. Anti-groups. J Protoalbumose (amplioalbTiinoae) Heteroalbumose (amphoalbumose) Denteroalbumose (amphoalbumose) Deuteroalbnmose (amphoalbumose) Amphopeptone Antialbumid Deuteroalbumose (antialbumose) Amphopeptone Antipeptone On the basis of these facts, and others not yet men- tioned, we may accept provisionally, at least, the above schematic view, suggested in part by Neumeister,' of the ' Zur Kentniss der Albumosen, Zeitsohr. f. Biol., Band 33, p. 391. 42 DIGESTIVE PROTEOLYSIS. general line of proteolysis as it occurs in pepsin-digestion ; a view which clearly expresses the significant relationship of the hemi- and anti-groups in the proteid molecule. The dark and light lines in this scheme are intended to represent the relative share which the hemi- and anti- groups take in the formation of the individual bodies. Thus, we see that protoproteoses have their origin mainly in the hemi-groups of the molecule, although, as the fine line indicates, anti-groups are somewhat concerned in their construction. Heteroproteoses, on the other hand, come mainly from the anti-groups, but still some hemi-groups have a part in their structure. As previously stated, these two primary proteoses by further hydrolytic action may be transformed into secondary products ; viz., into deutero- proteoses, but, as the above scheme indicates, the two deutero bodies will be more or less unlike in their inner nature. In one sense, they are both amphodeuteropro- teoses, but they necessarily differ in the proportion of hemi- and anti-groups they contain. By the still further action of pepsin-acid, the deutero bodies may be changed, in part at least, into peptone, *. e., into amphopeptone, although, as Neumeister has pointed out, protoproteose tends to yield an amphopeptone in which the hemi-groups predominate, while the peptone coming from heteropro- teose contains an excess of anti-groups. Moreover, in the gastric digestion of any simple proteid a certain number of anti-groups are split off in the form of antialbiimid, a body which is only slowly digestible in pepsin-acid. By the very powerful proteolytic action of a strong gastric juice, however, antialbumid may be somewhat digested, and is then transformed into antideuteroalbumose, which in turn may be eventually changed into antipeptone. From these statements it is evident that a given proteid exposed to pepsin-proteolysis may give rise to a large STETJCTUEE OF THE ALBUMIN MOLECULE. 43 number of products ; in fact, to a far larger number than is implied by the names in the above scheme. Thus, at first glance you would be inclined to say there can be only three deuteroalbumoses, for example ; one, a pure anti- body, the other two, amphoalbumoses, differing from each other simply in their content of hemi- and anti-groups. It must be remembered, however, that the inner constitution of these bodies, as implied by the relative proportion of the above groups, may vary to almost any extent. Thus, every variation in the number of anti-groups split ofE from the original albumin molecule to form antialbumid means just so much of a change in the relative ^proportion of hemi- and anti-groups entering into the structure of both primary and secondary albumoses. Hence, as you can see, digestive proteolysis, even in gastric digestion, is a some- what complex process. "We have to deal not only with a number of bodies superficially unlike, as the primary and secondary proteoses and peptones, but these bodies may show marked variations in structure dependent upon the exact conditions attending their formation. Evidently, the complexities attending digestive prote- olysis are connected primarily with the complex nature of the proteids themselves, while proteolysis, as a process, is made possible through the natural tendency of the proteids to undergo hydration and cleavage. LECTUKE 11. PEOTEOLYSIS BY PEPSIN-HYDEOCHLOEIO ACID, WITH A CON- SIDEEATION OF THE GENEEAL NATUEE OF PEOTEOSES AND PEPTONES. PEOTEOLYSIS BY PEPSIN-ACID. Gastric digestion is essentially an acid digestion. As a proteolytic agent, pepsin can act only in the presence of acid, and we have every reason for believing that the enzyme and the acid form a compound, which in turn com- bines with the proteid undergoing digestion ; or, what amounts to much the same thing, that the acid perhaps forms first a compound with the proteid, to which the pep- sin can then unite to form a still more complex compound capable of undergoing hydration and cleavage. Pepsin- proteolysis, therefore, is strictly the proteolysis produced by pepsin-acid. In view of this fact, we may well give a moment's thought to the nature and origin of this acid. Without attempting any statement of the gradual develop- ment of our knowledge regarding the acid of the gastric juice, we may accept the now well-established fact that the acid is hydrochloric acid, and that it has its origin in the parietal, or so-called border-cells of the gastric glands. That the acid is derived from the decomposition of chlo- rides is practically self-evident, but Cahn' has added experi- mental proof which removes all shadow of doubt, through his study of the gastric secretion in animals deprived for many days of salt ; the gastric juice in such cases being per- fectly neutral in reaction, but normal as regards its content of pepsin. ' Die Magenverdauung im ohlorhunger. Zeitsohr. f. physiol. Chem., Band 10, p. 532. FOKMATION OF HYDEOCHLOEIC ACID. 45 The way in wHcli the specific gland-cells manufacture free hydrochloric acid out of material contained in an alka^ line medium is somewhat doubtful. There are, however, at the present day two theories worthy of special notice. The first is based upon observations made by Maly' many years ago, which tend to show that certain mineral salts present in the blood are capable of reacting upon each other with formation of hydrochloric acid. Thus, while the blood is an alkaline fluid, it really owes its alkalinity to the presence of two acid salts, viz., sodium bicarbonate (HNaCOj) and disodium hydrogen phosphate (BLNa^PO,). This latter compound, acted upon by the carbonic acid of the blood, is transformed into a dihydrogen sodium phos- phate with simultaneous formation of acid sodium carbo- nate, as shown in the following equation : Na^HPOi + 002 + HjO = NaHjPGi + HNaOOs. This acid sodium phosphate dissolved in a fluid containing sodium chloride, gives rise to free hydrochloric acid by a very simple reaction : NaHjPO, + NaOl = Na^HPOi -I- HOI. It is also to be noted that the disodium hydrogen phos- phate, may, likewise, give rise to hydrochloric acid through its action on calcium chloride, as indicated by the following equation : 3 NaaHPOi + SOaCU = CasCPOi)! + 4NaCl + 3H01. It is thus evident that hydrochloric acid may originate in the inter-reaction of these several salts which are known to be present in the blood; but obviously, the above reac- tions cannot take place in the blood itself, and we must look to the selective power of the epithelial cells of the gastric glands, as suggested by Gamgee," for the with- ' Untersachimgeii uber die Quelle der Magensaf tsanre. Annalen d. Ohem. u. Pharm., Band 173, p. 237. * Physiological Chemistry of the Animal Body, vol. 3, p. 113. 46 DIGESTIVE PEOTEOLTSIS. drawal of the needed salts from the blood. Once present in the acid-forming cells, and perhaps aided by the inher- ent qualities of the protoplasm, the necessary chemical reactions may be assumed to take place, after which the newly formed acid may pass from the gland-cells into the secretion of the gland. A later theory regarding the formation of the acid of the gastric juice emanates from Liebermann.' This investi- gator claims the existence in the mucous membrane of the stomach of an acid-reacting, nuclein-like body, which is apparently a combiuation of the phosphorized substance lecithin with a proteid. To this compound body Lieber- mann gives the name of lecithalbumin. It is apparently located in the nuclei of the gastric cells, is strongly acid in reaction, and, according to Liebermann, is an important agent in the production of the free hydrochloric acid of the gastric juice, although its action is somewhat indirect. According to this theory, the free acid is formed in the mucous membrane of the stomach from sodium chloride, through the dissociating action of the carbonic acid coming from normal oxidation. The thus-formed acid then diffuses in both directions, viz., through the lumen of the gland into the stomach-cavity, and in part in the opposite direc- tion into the veins and lymphatics. It is the assumed function of the lecithalbumin to react with the alkaline sodium carbonate, produced simultaneously with the hydro- chloric acid. This naturally gives rise to the liberation of carbonic acid and to the formation of a non-diffusible- sodium-lecithalbumin compound, which is retained for the time being in the body of the cell. When the circulation of the blood, accelerated by the digestive process, returns to its ordinary pace, this latter compound is slowly decom- ' Studien iiber chemisolie Processe in der MagenschleimliaTit. Pfluger's Arohiv f. Physiol., Band 50, p. 25. Neue IJntersuchungen iiber das Lecithalbumin. Liebermann, Ibid. , Band 54, p. 573. FORMATION OF HTDEOOHLOKIO ACID. 47 posed by the carbonic acid with formation of the readily diffusible sodium carbonate, which passes into the blood- current. The rate of this latter reaction is impeded, or, perhaps regulated, by the swelling up of the lecithalbu- min-containing cells, thus rendering the imbibition of the carbonic acid a slow process. The rate of production of the hydrochloric acid by this hypothetical process depends primarily upon the blood supply, and the oxidative changes by which carbonic acid is formed. There is much that might be said for and against this theory,' but we cannot stop to discuss it here. Like the previous theory, it implies the production of hydrochloric acid from a chloride or chlorides, through chemical pro- cesses taking place in the stomach-mucosa, and presumably in the large border-cells of the peptic glands. This hydro- chloric acid, as you know, in the act of secretion, reacts upon the pepsinogen with which it may come in contact, transforming it into pepsin. It also has the power of com- bining with all forms of proteid matter, not excepting the products of proteolytic action, to form acid compounds in which the so-combined acid, although equal quantitatively to the original amount of free acid, is less active in many ways. Thus, it does not possess in the same degree a destructive action on the amylolytic ferments ; " it does not play the same part in aiding the proteolytic action of pep- sin, and its antiseptic power is far from equal to that of a like amount of free acid.' With relatively large amounts of proteid, we may have half or even quarter saturated proteid molecules, in which ' See discussion by P16sz and Liebermann in Jahresbericht fur Thier- chemie, Band 23, p. 360. * Chittenden and H. E. Smith : Studies in Physiol. Chem., Yale Uni- ver., vol. i., p. 18. ' Compare F. O. Cohn : Ueber die Einwirkung des kunstlichen Magensaftes auf Essigsaure- und Milchsauregahrong. Zeitsohr. f . physiol. Chem., Band 14, p. 74. 48 DIGESTIVE PEOTEOLTSIS. the weakness of the combined acid is far more pronounced than in the case of the fully saturated molecule. Such a condition of things must obviously exist in the early stages of gastric digestion. Witli an excess of proteid matter in the stomach, some time must elapse before the secretion of hydrochloric acid will be suflBcient to furnish acid for all of the proteid matter present, yet pepsin-prote- olysis does not wait the appearance of free acid. Indeed, the proteid matter may not have combined with more than half its complement of hydrochloric acid before digestive proteolysis is well under way. I have made many analy- ses of the stomach-contents after test meals, and under other conditions, where no free acid could be detected by the tropaeolin test, or better, by Giinzburg's reagent (phlo- roglucin-vanillin), although phenolphthalein as well as lit- mus showed strong acid reaction, and yet not only could acid-albumin be detected in the filtered fluid, but likewise proteoses and peptones. In other words, pepsin-proteolysis can proceed in the absence of free hydrochloric acid, although not at the same pace. Hence, proteoses and even peptones may make their appearance in the stomach-con- tents at a very early period of digestion, i. e., the final pro- ducts of proteolysis may be found in a mixture contain- ing even a large proportion of wholly unaltered pro- teid, and obvioiisly at an early stage in the process. Expressed in other language, a portion of the first formed acid-albumin or syntonin may be carried forward by the digestive process to the secondary proteose and peptone stage, before the larger portion of the ingested proteid food has even combined with sufficient acid to insure the com- plete formation of acid-albumin. This introduces another factor, to be referred to later on, viz., the relative combining power of different forms of proteid matter, especially the proteoses and peptones, as contrasted with native proteids. PEOTEOLT8I8 IN PRESENCE OF COMBINED ACID. 49 In proof of the statement that pepsin-proteolyeis can pro- ceed in the absence of free hydrochloric acid, provided com- bined acid be present, allow me to cite one or two experi- ments bearing on this point. A perfectly neutral solution of egg-albumen, containing 0.8169 gramme of ash-free albumin per 10 c.c. of fluid, was employed as the proteid material. In order to completely saturate the proteid con- tained in 20 c.c. of this neutral albumen solution, 50 c.c. of 0.2 per cent. HCl were required. Two mixtures were then prepared as follows : A. Twenty c.c. of the neutral albumen solution + 50 c.c. 0.2 per cent. HCl + 30 c.c. of a weak aqueous solution of pepsin, perfectly neutral to litmus. This mixture gave only the faintest tinge of a reaction for free acid when tested by Giinzburg's reagent. JB. Twenty c.c. of the neutral albumen solution + 25 c.c. 0.2 per cent. HCl -1- 30 c.c. of the neutral pepsin solution. In this mixture, the proteid matter was obviously only half saturated with acid. The two solutions were placed in a bath at 40° C, where they were allowed to remain for forty-four hours, a little thymol being added to guard against any possible putrefactive changes. At the end of this time the amount of undigested albumin was accurately determined. The 20 c.c. of original albumen solution contained 1.6338 grammes of dry coagulable albumin. At the end of the forty-four hours, A contained only 0.5430 gramme of unaltered albu- min, or acid-albumin, while £ contained 1.2225 grammes. That is to say, in the mixture A, where the acid existed wholly in the form of combined acid, but with the albumin completely saturated, 1.0908 grammes of the proteid were converted into soluble albumoses and peptones. In £, on the other hand, where the albumin was only half saturated with acid, 0.4113 gramme of the proteid was converted 5 60 DIGESTIVE PEOTEOLTSIS. into soluble products. This difference in action is made more striking by the statement that where the proteid was only half saturated with acid, 25.1 per cent, of the albumin was digested ; while with a complete saturation of the pro- teid, 66.7 per cent, of the albumin was digested. To give emphasis to this matter, a second experiment may be quoted as follows : The proteid used was the same neutral solution of egg-albumen containing 0.8169 gramme of albumin per 10 c.c. Two mixtures were pre- pared as follows : A. Ten c.c. of the neutral albumen solution + 21.7 c.c. 0.2 per cent. HCl, the amount needed to completely satu- rate the proteid, H- 40 c.c. of a weak solution of pepsin, perfectly neutral, B. Ten c.c. of the albumen solution + ]0.9 c.c. 0.2 per cent. HCl -F 40 c.c. of the pepsia solution, making a mix- ture half saturated with acid. These two solutions were warmed at 40° C. for seven- teen hours. The extent of digestive action was then deter- mined, when it was found that in A only 0.1638 gramme of the proteid was undigested, while in B, 0.6088 gramme remained unaltered. In other words, where the proteid was completely saturated with acid, but with an utter lack of free acid, 79.9 per cent, of the albumin was converted into albumoses and peptone, while in the mixture half satu- rated with acid only 25.4 per cent, was digested. These two experiments thus give striking proof that free acid is not absolutely essential for pepsin-proteolysis. Digestion is, to be sure, more rapid and complete when free hydrochloric acid is present, but proteolysis is still possible, and even vigorous, when there is a marked defici- ency of free acid. Further, as we have seen, proteolysis may proceed to a certain extent even though the amount of acid available is not sufficient to combine with more than half the proteid matter present. THE AFFINITY OF PEOTEIDS FOE ACID. 51 These facts at once raise the question whether the prod- ucts of proteolysis may not have a stronger affinity for acid than the native proteids ; an affinity so strong that they may be able to withdraw acid from the acid-albumin first formed. One of our conceptions regarding pepsin-proteolysis is that acid is necessary for every step in the proteolytic process. A primary albumose, for example, cannot be further changed by pepsin, unless there is acid present for it to combine with. This being true, it is clear, ia view of the fact that even peptones may appear in a digestive mixture containing an amount of acid insufficient to combine even with the albumin present, that the products of proteolysis must withdraw acid from the acid-albumin first formed. In regard to the first point, my own experiments certainly tend to show that the products of gastric digestion do com- bine with larger amounts of hydrochloric acid than undi- gested proteids ; and further, that of the several products of proteolysis, the secondary proteoses combine with a larger percentage of acid than the primary proteoses, while true peptones combine with still larger amounts. In other words, the simpler and more soluble the proteid, the larger the amount of acid it is capable of combining with ; a statement which accords with results obtained by other workers' in this direction. Further, another fac- tor of considerable importance in connection with the natural digestive process is that a dissolved proteid, such as protoalbumose for example, will combine more readily with free acid than an insoluble proteid ; from which Gillespie^ is led to infer that in pepsin-proteolysis where there is no free acid present, only acid-albumin, proteoses may be formed to a limited extent at the expense of some of the acid of the acid-albumin, a portion of the latter being ' See especially Gillespie : Gastric Digestion of Proteids. Journal of Anat. and Physiol.., vol. 27, p. 207. ^Loo. cit. 52 DIGESTIVE PEOTEOLTSis. perhaps reconverted into albumin. The ability of the pro- teoses, however, to withdraw acid from its combination with a native proteid is perhaps best indicated by Kossler's' experiments, which show that a solution of acid-albumin containing only enough hydrochloric acid to hold the albu- min dissolved, on being warmed at 40° C. for some hours with addition of a neutral solution of pepsin, may undergo partial conversion into albumose or peptone. In spite of these facts, there is some evidence that whCe proteoses and peptones have the power of combining with more acid than a like weight of native proteid, the latter, leaving out all action of the pepsin, has a stronger affinity for the acid ; in fact, the firmness or strength of the union appears to diminish as the products become simpler." Hence, a peptone separated from a digestive mixture, will part with its combined acid somewhat more readily than acid-albumin for example, although on this point there is not complete unanimity of opinion." In digestive prote- olysis, however, where the pepsin is accompanied by a minimal amount of hydrochloric acid, insufficient perhaps to even half saturate the proteid present, the formation of proteoses and peptones must be accompanied by a with- drawal of acid from its combination with the native proteid. In illustration of some of these points, and especially of the statement that the products of gastric digestion have the power of combining with more hydrochloric acid than the original proteid, allow me to cite the following experi- ment : 10 c.c. of a neutral solution of egg-albumen contain- ' Beitrage zur Methodik der quantitativen Salzsaurebestimnmng im Mageninlialt. Zeitsohr. f. physiol. Ohem., Band 17, p. 93. '' Compare Blum : Ueber die Salzsaurebindung bei klinstlioher Ver- dauung. Zeitsohr. f. klin. Medioin, Band 31, p. 558. ' See Sanson! : Beitrag zur kenntniss des Verhaltens der Salzsaure zu deH EiweisskSrpem in Bezug auf die Ohemisohe Untersuohung des Magensaftes. Berliner klin. Woobensohrift, 1893, Nos. 42 and 43. THE AFFINITY OF PEOTEIDS FOE ACID. 53 ing about 0.82 gramme of pure dry albumin, free from mineral salts, required 23.8 c.c. of 0.2 per cent, bydro- cbloric acid to completely saturate tbe proteid matter. A mixture was tben prepared as follows : 10 c.c. of tbe albumen + 24 c.c. 0.2 per cent. HCl + 30 c.c. of a neutral pepsin solution, tbe mixture sbowing a faint trace of free acid wben tested by Giinzburg's reagent. Tbis solution was placed in a tbermostat at 38° C, and from time to time a drop of tbe fluid was removed and tested for free acid. If no reaction could be obtained, 0.2 per cent, bydrocbloric acid was added to tbe mixture, until Giinz- burg's reagent sbowed free acid to be again present. Tbe following table sbows the rate of disappearance of free acid, and tbe amounts of 0.2 per cent. HCl required to make good tbe deficiency. The mixture was placed at 38° C. on February 6tb, at 11.30 a.m., and, as stated, con- tained a trace of free acid, 24 c.c. 0.2 per cent. HCl having been added to accomplish this result. Time. Acid add! ed to Bhow trace of free Februarys, 11.30 a.m. 3.15 p.m. 4.5 O.C, ,0.3 per cent. HCl. " 5.00 p.m. 1.0 " It ti it February 7, 8.45 a. m. 8.0 " (C a " 3.00 p. M. 1.0 " ii (( (( " 5.00 p.m. 1.5 " it ii it February 8, 8.30 a. m. 1.0 " ti 11 (( " 3.30 P.M. 0.0 " (( u February 9, 8.30 a.m. 3.0 " (t ii it February 10, 9.30 A. M. 3.0 " ct 11 17.0 From these results several interesting conclusions may be drawn, in conformity with tbe statements already made. Thus, as soon as proteolysis commences, tbe products formed begin to show their greater affinity for acid by withdrawing acid from its combination with tbe native 54 DIGESTIVE PEOTEOLYSIS. proteid, a supposition wtich is necessary to account for even the starting of the proteolytic process. Further, it is evident that proteoses and peptones combine with a far larger equivalent of acid than the native albumin is capa- ble of ; 17 c.c. of 0.2 per cent. HCl being required in the above experiment to satisfy the greater combining power of the newly formed products. This doubtless depends upon the cleavage of the large proteid molecule into a number of smaller or simpler molecules, each of the latter, perhaps, combining with a like number of HCl molecules. This view of the relationship of the individual proteoses and peptones is one more or less generally held, and is sup- ported by many facts.' However this may be, it is evident that the products of pepsin-proteolysis combine with a larger amount of hydrochloric acid than the mother-pro- teid, and that the transformation of the latter, at least under the conditions of this experiment, is a slow and gradual process. In the living stomach, on the other hand, where the secretion of acid is progressing with ever- increasing rapidity, it is easy to see that the process of proteolysis would naturally be much more rapid. Just here we may recall the theory advanced by Eichet" quite a number of years ago that the acid of the gastric juice is a conjugate acid, composed of leucin and hydro- chloric acid, a theory which has found little acceptance. Klemperer,' however, assumed that solutions of leucin hydrochloride with pepsin would not digest albumin, but Salkowski and Kumagawa* have shown by experiments that leucin and other amido-acids, as glycocoU, may be dis- ' See Gillespie : On the Gastric Digestion of Proteids. Journal of Anatomy and Physiology, vol. 27, p. 309. ' Le sue gastrique chez I'honime et les animaux, ses propri^t^s cMm- iques et biologiques. Paris, 1878. ' Zeitsohr. f . Win. Medicin, Band 14, Heft 1 and 3. * Ueber den BegrifE der f reien und gebundenen SalzsSure im Magensaf t. Virchow's Arohiv, Band 132, p. 235. PEOTEOLTSIS IN THE PEBSENCE OF AMIDO-ACIDS. 55 solved in hydrochloric acid in such proportion that the solution is practically composed of leucin hydrochloride, without interfering with the digestive action of pepsin-acid on blood-fibrin ; the solution being physiologically active, although Giinzburg's reagent shows an entirely negative result for free acid. If the matter is studied quantita- tively, however, it will be found that the amido-acids combining in this manner with the hydrochloric acid of the gastric juice do give rise to some disturbance of prote- olytic action ; ' *. e., digestion may be less rapid, especially on egg-albumin, a conclusion which Salkowski" has lately confirmed. Still, under such circumstances, digestion does go on and at a fairly rapid rate ; hence, if there is a com- bination between the acid and these organic bodies, as is indicated by Giinzburg's reagent, the acid is still active physiologically, even more so than in the compound formed by the interaction of proteid and acid. In other words, many of these neutral organic bodies that may originate in the stomach through fermentative processes, or otherwise, and which tend to combine with the acid of the gastric juice, do not, as a rule, impede pepsin-proteolysis to the same extent that an excess of proteid matter may. In fact, in artificial digestions long continued, pepsin-acid solutions containing considerable leucin, for example, may accomplish as much in the way of digesting proteid matter as the same amount of pepsin-acid without leucin ; but the inhibitory action of the amido-acid is there, and may be shown during the first few hours of the experiment, when less proteoses and peptones are formed than in the control experiment without leucin. It is foreign to our subject to discuss here methods for > Eosenheim : Centralbl. f. klin. Medicin, 1891, No. 39. F. A. Hof- man, ibid., No. 43. ' XJeber die Bindung der Salzsatire duroli Amidosauien. Virohow's Arcbiv, Band 137, p. 501. 56 DIGESTIVE PROTEOLYSIS. the detection of so-called free and combined hydrocUoric acid in the stomach-contents, or the special significance of such findings in health and disease. I cannot refrain, however, in connection with what has been said above concerning the proteolytic action of pepsin in the presence of combined acid, from saying a word concerning the usual deductions drawn from the absence of free acid in the stomach-contents. As Langermann' has recently expressed it, we have methods for discriminating between free and combined acid ; we can, moreover, determine the amount of free acid, but is it not equally important to be able to say something definite concerning the amount of combined acid in the stomach-contents? Even in the absence of free hydrochloric acid there may be a sufficient amount of HCl secreted to answer all the purposes of digestion, and yet at no time may there be any free acid present to be detected by the various color-tests ordinarily made use of. I am aware that in ordinary examinations of the stomach -eon tents after a test meal the results are essentially comparative, and possibly all that are necessary for clinical purposes. What I wish to emphasize, how- ever, is that in order to pass conclusively upon the suf- ficiency or insufficiency of the gastric secretion, it is wise to know not only the total acidity of the stomach contents and whether there is free acid or not, but to know more about the amount of combined acid present. Thus, there is a natural tendency to divide the fluids withdrawn from the stomach into three groups, viz., those which contain free acid in moderate amount, those which contain free acid in excess, and those in which free acid is entirely absent ; but in the latter group, there may be very marked differences in the amount of acid combined with the proteid and other material present. It appears to me that one of the ques- ' Virohow's Arohiv, Band 128, p 408. IMPORTANCE OF COMBINED ACID. 57 tions to be answered is whether there is sufficient combined HCl present to meet all the requirements for digestion. If there is, that gastric juice may be just as normal as the one containing free mineral acid, and yet, according to our present tendencies, we should be inclined to call the juice containing no free acid abnormal, although there may be sufficient combined acid present to meet all the require- ments for digestion. Hence, in examination of the stomach- contents, it is well to consider the use of those methods which tend to throw light upon the amount of combined acid present, for in my opinion it is only by a determina- tion of the total amount of combined acid that we can arrive at a true estimate of the extent of the HCl deficiency. Obviously, in simple clinical examinations of the stomach-contents after a test meal, where proteid matter is not present in large amount, free acid may rea- sonably be expected to appear after a definite period ; but in any event, it is well to remember that free hydrochloric acid is not absolutely indispensable for fairly vigorous pro- teolytic action, and that in the presence of moderate amounts of proteid matter a large quantity of acid is required to even saturate the albuminous material. Consider for a moment the amount of acid a given weight of proteid will combine with, before a reaction for free acid can be obtained. Thus, Blum' has stated that 100 grammes of dry fibrin will require 9.1 litres of 0.1 per cent, hydrochloric acid to completely saturate it. Hence, with a daily consumption of 100 grammes of proteid, there would be needed for gastric digestion 4.5 litres of 0.2 per cent, hydrochloric acid daily, and even this would not suffice to give any free acid, assuming that none of the acid is used over again. The results I have already given for egg-albumin tend to show that 1 gramme of pure 1 Zeitschr. f. klin. Medioin, Band 21, p. 558. 58 DIGESTIVE PEOTEOLTSIS. .albumin, free from inorganic salts, when dissolved in a moderate amount of water will combine with about 30 c.c. of 0.2 per cent, hydrochloric acid. Consequently, on this basis, 100 grammes of dry egg-albumin will combine with 3 litres of 0.2 per cent. HCl, and not until this amount of acid has been added to such a mixture will reaction for free acid be obtained with Giinzburg's reagent. Hence we can easily see, in view of these figures, that the produc- tion of hydrochloric acid by the gastric glands may at times be very extensive, without the stomach-contents necessarily containing free acid. While I am by no means willing to agree with Bunge ' "that the chief importance of the acid of the gastric juice is its action as an antiseptic, I am decidedly of the opinion that the lack of free hydrochloric acid in the stomach-con- tents is more liable to cause disturbance through the con- sequent unchecked development of bacteria than through lack of proteolytic action, assuming, of course, the presence of a reasonable amount of combined HCl. The hydro- chloric acid of the gastric juice unquestionably plays a very important part in checking the growth and develop- ment of many pathogenic bacteria, as well as of less poisonous organisms, which are taken into the mouth with the food. On all, or at least on nearly all of these organ- isms, hydrochloric acid exerts a far greater destructive .action when free than when combined with proteid matter. As Cohn " has plainly shown, both hydrochloric acid and pepsin-hydrochloric acid quickly hinder acetic- and lactic- acid fermentation, but when the acid is combined with peptone, for example, it is no longer able to exercise the same inhibitory influence. It is also important to note ' Physiologisohe und Pathologisohe Chemie, p. 153. ' Ueber die Einwirkung des kiinstliohen Magensaf tea auf EssigsSure- Tmd Milchsaure gShrung. Zeitschr. f . physiol. Chemie, Band 14, p. 75. See also Hirsclifeld ; Pfltiger's Arohiv f. Physiol., Band 47, p. 510. AXTISEPnC ACTIO:^ OP FBZE ACXD. 59 that the lactic-acid fennent is not so sensitive to hydro- chloric acid as the acetic-acid ferment. ConsequentlT. when lactic-acid fermentation is once developed a compara- tively large amount of HCl is required to arrest it. Hence, as vre all know, a diminished secretion of hydro- chloric acid renders possible acid fermentation of the stomach-contents, as well as putrefactive changes which would not occur in the presence of free HCl, and which are very incompletely checked when the acid is over- saturated with proteid matter. Pepsin-proteolysis, however, may proceed, to some extent, at least, even though a small amount only of com- bined acid is present. The combined acid, however, must be hydrochloric acid, if proteolysis is to be at all marked. To be sure, pepsin wiU act in the presence of lactic acid, as well as in the presence of other organic acids, and inor- ganic acids, likewise, but such action at the best is consid- erably weaker than the action of pepsin-hydrochloric acid.' The ferment pepsin can exert its maximum action only in the presence of free hydrochloric acid. There must be sufficient HCl to combine with all of the proteid matter present, and the products of proteolysis as fast as they are formed, if digestion is to be rapid and attended with the formation of a large proportion of the final products of proteolysis. It is under such conditions that our study of pepsin-proteolysis is usually conducted. Further, it is to be remembered that our knowledge of the products of such proteolytic action depends almost entirely upon data accumulated by artificial digestive experiments. In no other way can we be absolutely certain of the conditions under which the proteolysis is accomplished, for it is a significant fact, perhaps plainly evident from what has ' Chittenden and Allen : Tnflnence of Taiioas Inoiganic and Alka^ loidal Salts on the Proteolytic Action of Pepsin-HydrochlOTic Acid. Stndies in PhysioL Caiem., Yale University, toL 1, pp. 91, 94. 60 DIGESTIVE PEOTEOLT8I8. already been said in the preceding lecture, that the charac- ter of the products resulting from ordinary proteolysis is dependent in great part upon the attendant circumstances. Thus, with a relatively small amount of acid, and perhaps also of pepsin, the initial products of proteolysis are espe- cially prominent, while with an abundance of both pepsin and free acid, coupled with long-continued action, the final products predominate. Between these two extremes there are many possible variations, as was, I think, made clear in the previous lecture. At the same time, it is to be noticed that these differences are mainly differences in the propor- tion of the several products, rather than in the nature of the resultant bodies. In a general way, the products of pepsin-proteolysis may be divided into three main groups, viz., bodies precipi- tated by neutralization and represented mainly by the so-called syntonin or acid-albumin ; bodies precipitated by saturation of the neutralized fluid with ammonium sulphate and represented by proteoses ; bodies non-precipitable by saturation with ammonium sulphate and represented by amphopeptones. The relationship of the individual prod- ucts may be clearly seen from the following scheme, arranged after the plan suggested by Neumeister. Native Protbid. Syntonin. Protoproteose. Heteroproteose. (dysproteose). Deuteroproteose. Deuteroproteose. Peptone. Peptone. DETECTION OF THE PEODTTCTS OF DIGESTION. 61 It is, of course, to be understood that this is not intended to represent anything more than the order of formation of the several bodies, no attention being paid here to the hemi- or anti-character of the several products, or classes of products. Thus, proto and heteroproteose are primary bodies formed directly from the initial product syntonin by the further action of the ferment. In the same sense, deuteroproteose is a secondary proteose, being formed by the further hydration of the primary body. Lastly, pep- tones, the final products of pepsin-proteolysis, are the result of the hydration and possible cleavage of deuteroproteoses. Further, in almost every gastric digestion there is also formed a small amount of antialburaid, a product insoluble in dilute hydrochloric acid and which consequently appears as an insoluble residue. This body is very resistant to the action of pepsin-acid when once formed, but may be slowly converted, in part at least, into a soluble antialbumose and thence into antipeptone. All of these bodies can be readily identified in any digestive mixture containing them by a few simple reac- tions. Thus, after having removed any acid-albumin or syntonin present by neutralization, the concentrated fluid can be tested at once.- If primary proteoses are present, the neutral fluid will yield a more or less heavy precipitate on addition of crystals of rock-salt, precipitation being com- plete only when the fluid is saturated with the salt. Further, if the proteoses are present in not too small quan- tity, nitric acid added drop by drop to the neutralized fluid will produce a white precipitate, readily soluble on applica- tion of heat but reappearing as the solution cools. If primary proteoses are wholly wanting, then no precipitate will be obtained by acid unless the fluid is saturated with salt, in which case a portion of the deuteroproteose will be precipitated. The two primary proteoses differ from each 62 DIGESTIVE PEOTEOLTSIS. other especially in solubility ; protoproteose being readily soluble in water alone, while heteroproteose is soluble only in salt solutions, dilute acids, and alkalies. Hence, when these two bodies are precipitated together by saturation with salt, they may be readily separated by dissolving them in a little dilute salt solution, anddialyzing the fluid in run- ning water until the salt is entirely removed ; heteroproteose win then be precipitated, while the proto-body remains in solution. By long contact with water, and even with concentrated salt solutions, heteroproteose tends to undergo change into a semi-coagulated form, named dysproteose, insoluble in dilute sodium-chloride solutions. This body can be recon- verted into heteroproteose, in part at least, by solution in dilute acid, or alkali, and reprecipitation by neutralization. As a class, the proteoses are characterized by far readier solubility in water than native proteids, by a far greater degree of diffusibility, by non-coagulability by heat and by alcohol, although precipitable by the latter agent. Further, nearly all proteose precipitates are exceedingly sensitive toward heat, tending to dissolve as the fluid is warmed and reappearing as the solution cools. In fact, this peculiarity often serves as a means of identification. Potassium fer- rocyanide and acetic acid, picric acid in excess, and likewise cupric sulphate, all precipitate the primary proteoses, while deuteroproteose is only slightly affected by these reagents, or indeed not at all. In order to separate the secondary proteose from the primary bodies in the absence of peptones, the fluid is neu- tralized as nearly as possible, and then, after suitable con- centration, is saturated with sodium chloride for the partial precipitation of the primary proteoses. To the clear filtrate, acetic acid' is added drop by drop as long as a precipitate > Saturated with sodium chloride. SEPAEATION OF PEOTEOSES. 63 results, the latter being composed of a mixture of proto- proteose and deuteroproteose. That is to say, protoproteoses are not completely precipitated from neutral solutions by saturation with salt alone ; a little acid is required to com- plete it, but this tends to bring down a certain amount of deuteroproleose. From this filtrate, however, the deutero- body can be separated in a pure form by dialyzing away" the salt and acid, and then concentrating the fluid and pre- cipitating with alcohol. When the proteoses are mixed with peptones, the former must first be separated collect- ively by saturation of the fluid with ammonium sulphate. Peptones are especially characterized by non-precipi- tation with the ordinary precipitants for proteid bodies, and especially by the fact that they are wholly indifferent to saturation with ammonium sulphate either in neutral, acid, or alkaline fluids. This reaction, which constitutes the main, and perhaps the only absolute method of separat- ing peptones from proteoses must be carried out with great thoroughness in order to insure a complete precipitation of deuteroproteose. The latter stands midway between pri- mary proteoses and peptones in many respects, and seems to share with peptones something of a tendency to resist pre- cipitation by the ammonium salt. Indeed, as Kuhne' has recently pointed out, the last traces of deuteroproteose can be precipitated from the fluid only by long continued boil- ing of the ammonium sulphate-saturated fluid, and even then it is seldom complete unless the reaction of the fluid is alternately made neutral, acid, and akaline, and the heat- ing continued for some time after each change in reaction. Under such circumstances, the last portions of deuteropro- teose separate from the salt-saturated fluid and float on the surface in the form of an oily or gummy mass, while the •Erfahrungen iiber Albumosen und Peptone. Zeitschr. f. Biol., Band 39, p. 3. 64 DIGESTIVE PE0TE0LYSI8. true peptone remains in the fluid absolutely non-precipita- ble by the salt. In this filtrate, peptone can be detected by adding to a small portion of the fluid a very large excess of a strong solution of potassium hydroxide, followed by the addition of a few drops of a very dilute solution of cupric sulphate. If peptone is present a bright red color will appear, the intensity of which, with the proper amount of cupric sul- phate, will be proportional to the amount of peptone pres- ent. If it is desired to separate the peptone from the ammonium-sulphate-saturated fluid, there are several methods available, of which the following is perhaps the most satisfactory : The fluid is concentrated somewhat, and set aside in a cool place for crystallization of a portion of the ammonium salt. The fluid is then mixed with about one-fifth its volume of alcohol, and allowed to stand for some time, when it separates into two layers — an upper one, rich in alcohol, and a lower one, rich in salts. The latter is again treated with alcohol, by which another separation of the same order is accomplished. Finally, the lighter alcoholic layers containing the peptone are united, and exposed to a low temperature until consid- erable of the contained salt crystallizes out. The fluid is then concentrated, and after addition of a little water is boiled with barium carbonate until the fluid is entirely free from ammonium sulphate. Any excess of baryta in the filtrate is removed by cautious addition of dilute sulphuric acid, after which the concentrated fluid, reduced almost to a sirupy mass, is poured into absolute alcohol for precipitation of the peptone. So separated, the peptone formed in gastric digestion is exceedingly gummy, but can be transformed into a yellow- ish powder, very hygroscopic, of more or less bitter taste, and, when thoroughly dry, dissolving in water with a hiss- PEOPEPTONE A MIXTUEE OF PEOTEOSES. 65 ing sound and with considerable development of heat, like phosphoric anhydride.' I have introduced these dry chemical facts, none of which are especially new, because I deem them of consid- erable importance and because they are not very generally known. In fact, there seems to be a tendency on the part of some who are more or less familiar with the advances made iu our knowledge of the products of pepsin-proteoly- sis to question the existence of these different bodies, or to show at least a spirit of indifference toward these recent facts which have been gradually accumulated, and I may say accumulated at the expense of considerable labor. The time is past for calling the products of gastric digestion peptones ; it is time for a full recognition of the fact that pepsin-proteolysis is synonymous with the production of a row of bodies, chemically and physiologically distinct from each other, each endowed with individuality enough to admit of certain detection, and all bearing a certain specific and harmonious relationship to their neighbors, the other members of the series. Further, it is not enough to admit the formation of a sin- gle intermediate body, midway between syntonin and pep- tone. The so-called propeptone of the past is simply a mix- ture of proteoses, of ever changing composition, varying with each change in the proportion of the component pro- teoses. Each of these proteoses can be detected, under suitable conditions, in the products of every artificial diges- tion as well as in the stomach-contents, and no better measure of the proteolytic power of the natural stomach- secretion can be devised than a study of the character of the individual bodies present in the stomach-contents after a suitable test meal. The proper tests and separations can 'Kiilme and Chittenden: Peptones. Studies in Physiol. Chem., Yale University, vol. 3, p. 14. 6 66 DIGESTIVE PEOTEOLTSIS. be made with a small amount of tlie filtered fluid, and much light thrown upon the digestive power of the secretion by even a rough estimate of the proportion of primary and secondary proteoses and peptones formed in a given time, after the ingestion of a certain amount of proteid food. In pepsin-proteolysis we have to deal, in my opinion, with a series of progressive hydrolytic changes in which peptones are the final products of the transformation. Commencing with the formation of acid-albumin or synto- nin, hydrolysis and cleavage proceed hand in hand, under the guiding influence of the proteolytic enzyme, and each onward step in the process is marked by the appearance of a new body corresponding to the extent of the hydrolysis ; each body, perhaps, being represented by a row or series of isomers, all externally alike, but different in their inner structure, according to the proportion of hemi- and anti- groups contained in the molecule. As opposed to this theory, we have the older views of Maly," Herth,' Hennin- ger° and others, based upon observations which tend to show that peptones do not differ in chemical composition from the proteids which yield them. As a matter of fact, the products then analyzed were not peptones at all ; they were merely the primary products of pepsin-proteolysis, i. e., what we now term primary proteoses, and it is time we stopped using such data to enforce the theory that peptones are polymers of the proteids from which they are derived. In 1886, the writer, in conjunction with Professor Klihne, commenced a study of the various cleavage prod- ucts* formed by the action of pepsin-hydrochloric acid 1 Ueber die chemisohe Zusammensetstung und physiologiBohe Bedeu- tung der Peptone. Pfliiger's Archiv f. Physiol., Band 9, p. 585. ' Ueber die chemisobe Natur des Peptones nnd sein Verhaltniss zum Eiweiss. Zeitsohr. f. physiol. chem., Band 1, p. 377. 'De la Nature et du r61e pbysiologique des peptones. Paris, 1878. ■• Globulin and Globuloses. Studies in Physiol. Chem. Yale Univer- sity, vol. ii., p. 1. COMPOSITION OF PEOTEOSES AND PEPTONES. 67 from the better characterized and purer proteids, this being a continuation of our earlier work on the proteoses and peptones formed from blood-fibrin, serum-albumin, etc. This work I have continued in my laboratory up to the present time, with many co-workers, and as a result we have to-day a series of observations gradually accumulated during these last seven years, some the results of work car- ried on this last year, which speak in no uncertain way of the character of both the primary and secondary products of pepsin-proteolysis. Furthermore, in attempting to settle this question once for all, I have selected for study exam- ples from the various classes of both animal and vegetable proteids ; and as representatives of the latter liave had car- ried out two lengthy series of experiments on the crystal- lized proteids which occur so abundantly in some seeds, on the assumption that these crystalline bodies would furnish a certain guarantee of purity which might naturally be lacking in the amorphous proteids of animal origin. Some of these results are now placed together in the following tables, a study of which reveals some very interesting facts : COMPOSITION OF PEOTBOLTTIC PEODTJOTS FOEMED BY PEPSIN-HYDEOCHLOEIC ACID. Proteolysis of Blood-fibrin. Mother Proteid. ProtoflbilnoBe. i Heteroflbrin- ose.i Deuteroflbrin- oae.i Amphopep- tone.3 n 53.68 51.50 50.74 50.47 48.75 H 6.83 6.80 6.72 6.81 7.21 N" 16.91 17.13 17.14 17.20 16.26 R 1.10 0.94 1.16 0.87 0.77 23.48 23.63 34.24 24.65 37.01 ' Kuhne and CMttenden : ' Kuhne and Chittenden : vol. ii., p. 40. Zeitsohr. f. Biol., Band 30, p. 40. Studies in Physiol. Chem., Yale Univer., 68 DIGESTIVE PROTEOLYSIS. Proteolysis of Paraglohulin. ' Mother Proteia. Pi'otoglobulose. Heteroglobulose. Denteroglobiilose. c H N S 52.71 7.01 15.85 1.11 ) 23.24 f 51.57 6.98 16.09 25.36 52.10 6.98 16.08 24.84 51.52 6.95 15.94 25.59 Proteolysis of Coagulated Egg-albumin. Mother Proteid. Protoalhu- mose.s Heteroalbu- mnee.a Deuteroalbn- mose.^ Hemipeptone.9 C H N S 52.33 6.98 15.84 1.81 33.04 51.44 7.10 16.18 2.00 33.28 52.06 6.95 15.55 1.63 33.81 51.19 6.94 15.77 2.03 24.08 49.38 6.81 15.07 1.10 27.64 Proteolysis of Casein from Milk. Mother Proteid. Protocaseose.* Heterocae- eose.s a Deuterocas- eoBe.4 /3 Denterocas- eose.* H N S 53.30 7.07 15.91 0.82 I 22.03 ) 54.58 7.10 15.80 32.52 53.88 7.27 15.67 23.18 52.10 6.98 15.51 35.46 47.73 6.73 15.97 39.58 Proteolysis of Myosin from. Muscle.^ Mother Proteid. Protomyosinoae. DeuteromyoBiuoae. c 53.83 52.43 50.97 H 7.11 7.17 7.43 N 16.77 16.93 17.00 S 1.27 1.32 1.32 21.90 23.16 33.39 'Kiiline and Chittenden : Studies in Physiol. Ohem., YaJe Univer., vol. ii, p. 13. ' Chittenden and Bolton ; Ibid., vol. ii, p. 153. 'Kuhne and Chittenden : Zeitsohr. f. Biol., Band 19, p. 201. ^Chittenden : Studies in Physiol. Chem., Yale Univer., vol. iii., p. 80. ' Chittenden and Painter : Ibid., vol. ii., p. 195. ' Kiihne and Chittenden : Ibid. , vol. iii, p. 147. COMPOSITION OF PEOTEOSES AND PEPTONES. 69 Proteolysis of Elastin.' Mother Proteid. Protoelastose. DeuteroelastOBe. H N ) 54.34 7.37 16.70 31.79 54.53 7.01 16.96 31.51 53.11 7.08 16.85 33.96 Proteolysis of Gelatin.' Mother Proteid. Protogelatose. Deuterogelatose. 49.38 49.98 49.33 H 6.81 6.78 6.84 N 17.97 17.86 17.40 S 0.71 0.53 0.51 35.18 34.86 36.03 Proteolysis of Phytovitellin^ (" Crystallized J from Squash Seed. Mother Proteid. ProcoTltelloBe. Deuterovltellose. c 51.60 51.53 49.37 H 6.97 6.98 6.70 N 18.80 18.67 18.78 S 1.01 31.63 \ 23.83 25.35 Proteolysis of Phytovitellin " C Crystallized J from, Hemp Seed. Mother Proteid. Protovitelloae. Deuterovitellose, Peptone. c 51.63 51.55 49.78 49.40 H 6.90 6.73 6.73 6.77 N 18.78 18.90 17.97 18.40 S 0.90 1.09 1.08 0.49 31.79 31.73 34.44 34.94 ' Chittenden and Hart : Studies in Physiol. Ohem., Yale Univer., vol. iii, p. 37. - Chittenden and SoUey : Journal of Physiol., vol. xii, p. 33. ' Chittenden and Hartwell : Ibid., vol. xi, p. 441. ■* Chittenden and Mendel: Ibid., vol. xvii, p. 48. 70 DIGESTIVE PBOTEOLYSIS. Proteolysis of Glutenin^ from Wheat. Mother Proteid. ProtoglutenoBe. Beteroglntenose. Deuteroglntenose. c H N S 52.34 6.83 17.49 1.08 33.26 51.43 6.70 17.56 1.34 33.98 51.83 6.79 17.43 1.59 33.37 49.85 6.69 17.57 0.80 35.09 Proteolysis of Zein.' Mother Proteid. ProtozeosD. Deuteiozeose. c 55.38 53.39 51.31 H 7.36 6.87 6.88 N 16.13 16.10 16.27 S 0.60 1.54 1.08 20.78 32.20 34.46 In considering these results, it is to be noticed that there is a general unanimity of agreement except in the case of the albuminoid gelatin. In the proteolysis of this body, for some reason not explainable, the digestive products show no marked deviation from the composition of the mother-proteid, but in every other instance there is to be traced a distinct tendency toward diminution in the con- tent of carbon, proportional to the extent of proteolysis. In the primary bodies, proto and heteroproteoses, the per- centage of carbon is only slightly lowered; indeed, in some few cases, notably in elastin and casein, the primary products show a slight increase in their content of carbon, but in most instances there is a slight falling off in the percentage of this element. In the deuteroproteoses, how- ever, the loss of carbon is very marked. The percentage loss, to be sure, varies with the different proteids, doubtless ' Formerly called gluten-casein, and the products gluten-oaseoses. Chittenden and E. E. Smith ; Journal of Physiol., vol. xi, p. 430. ' Chittenden and WiUiams : Not heretofore published. PEOTEOLYSIS A HYDEOLTTIC PEOCESS. Tl dependent in part upon the nature of the proteid itself, and also, I think, upon the strength of the proteolytic agent employed and the duration of the proteolysis. It is to be further noticed that peptones, whenever analyzed, show a still further loss of carbon and also a marked loss of sulphur. In nitrogen there is no constant difEerence. On the assumption that these various products of pro- teolysis are formed by a series of hydrolytic changes, accompanied by cleavage of the molecule, we might at first glance look for a marked increase in the content of hydro- gen. But when we consider the size of the proteid mole- cule, with the small proportion of hydrogen contained therein and the large amount of carbon, it is plain that hydrolytic cleavage might naturally leave its mark on the percentage of carbon, rather than on the percentage of hydrogen of the resultant products. In view of these facts, the above results show nothing inconsistent with the theory that pepsin-proteolysis, as a rule, is accompanied by a series of progressive hydrolytic cleavages in which the primary proteoses are the result of a slight hydration, these bodies by continued proteolysis being further hydrated with formation of secondary proteoses, which in turn undergo final hydration and cleavage into true peptones. In accord with this theory, true peptones always show a marked dif- ference in composition from that of the mother-proteid, the most striking feature being the greatly diminished con- tent of carbon, which may be taken as a measure, in part at least, of the extent of the hydrolytic change. And it is to be noticed that the crystallized phytovitellins are no exception to the general rule ; the secondary vitelloses and peptones resulting from proteolysis bear essentially the same relationship to the mother-proteids that the albumoses from egg-albumin do. Moreover, the alcohol-soluble pro- teids, of which the zein of cornmeal is a good example, 72 DIGESTIVE PEOTEOLYSIS. show the same general tendency, and it is an interesting fact that the proteoses, or more specifically the zeoses, formed from this peculiar proteid, are readily soluble in water and show the general proteose reactions. It may also be mentioned that these zeoses, as well as the elastoses, are very resistant to further hydrolysis by pepsin-acid, and yield only comparatively small amounts of true peptones. In connection with this question of the composition of proteoses and peptones as formed by pepsin-proteolysis, it is interesting to note a recent observation recorded by Schiitzenberger.' This experimenter took 350 grammes of moist blood-fibrin, corresponding to 75.5 grammes of dry substance, and subjected it to proteolysis with 2.5 litres of a very strong pepsin-hydrochloric acid solution for five days. The resultant fluid was then freed from acid by treatment with silver oxide, after which the solution was evaporated to dryness on a water-bath and the residue dried in vacuo. This residue, termed by Schiitzenberger fibrin- peptone, was found on analysis to contain 49.18 per cent, of carbon, 7.09 per cent, of hydrogen, and 16.33 per cent, of nitrogen, thus agreeing very closely with true fibrin- peptone as analyzed by Kiihne and myself. Further, Schiitzenberger showed that the fibrin in undergoing this transformation had taken on 3.97 per cent, of water. But to my mind, the most significant fact connected with this experiment is the positive evidence it afEords, not only of hydration as a feature of peptonization by pepsin-acid, but that this greatly diminished content of carbon, so charac- teristic of peptones, and to a less extent of deuteroproteoses, is wholly independent of the methods of separation and purification ordinarily made use of. Thus, Schiitzenberger, in the above experiment, did not attempt any separation of ^Eecherohes but la constitution chimique des peptones. Comptes EenduB, vol. 115, p. 208. PEPTONIZATION NBTEE COMPLETE. 73 individual bodies. Proteolysis was carried out under con- ditions favoring maximum conversion into peptone, and tlie resultant product, or products, was analyzed directly without recourse to any methods of precipitation or puri- fication. To be sure, the substance analyzed could not have been peptone entirely free from proteose, but in any event it represented the terminal products of pepsin-pro- teolysis, and like true amphopeptone contained 3.5 per cent, less carbon than the original fibrin. Hence, we may conclude, without further argument, that peptonization in gastric digestion is the result of distinct hydrolytic action, in which the original proteid molecule is gradually broken down, or split apart, into a number of simpler molecules, the proteoses and peptones. Peptones, *'. e., amphopeptones, are the iinal products of gastric digestion; but to how great an extent is actual peptonization carried on in pepsin-proteolysis ? As we have seen, syntonin, primary proteoses, secondary prote- oses, and peptones are all products of pepsin-digestion, and it might perhaps be assumed that ultimately all of a given proteid undergoing pepsin-proteolysis would be converted into amphopeptone. Examination, however, shows that such is not the case, at least in artificial digestive experi- ments. Peptones are truly formed, and many times in large amount, but never under any circumstances have I been able to effect a complete transformation of any pro- teid into true peptone by pepsin-proteolysis ; there is always found a certain amount of proteoses more or less resistant to the further action of the ferment. Obviously, the nature and proportion of the individual products formed in any digestive experiment are dependent greatly upon the attendant conditions; but even with a large amount of active ferment, an abundance of free hydro- chloric acid, a proper temperature, and a long-continued Y4 DIGESTIVE PEOTEOLT8I8. period of digestion, even five and six days, there is never found a complete conversion into peptone. Indeed, the largest yield of peptone I have ever obtained in an artificial digestion is sixty per cent., while the average of a large number of results under most favorable circumstances is somewhat less than fifty per cent.' We understand that peptones are the products of the hydration and cleavage of previously formed proteoses. The primary proteoses pass into secondary proteoses and these into peptones, but for some reason this transforma- tion after a time becomes a slow and gradual process. At first there is a marked and rapid progression ; the proteid undergoing proteolysis is rapidly dissolved, and both pro- teoses and peptones may be detected in abundance. But if we continue to watch the changing relations of primary and secondary proteoses and peptones, we find that pro- gression soon ceases to be rapid, and eventually travels onward at a snail's pace. Thus, in one experiment with coagulated egg-albumin, there was found at the end of forty-eight hours' digestion with pepsin-hydrochloric acid, only thirty-seven per cent, of peptones with fifty-eight per cent, of proteoses, and yet digestion had been sufficiently vigorous to allow of a complete solution of the proteid in two hours. At the end of seventy-two hours the amount of peptones had increased to about forty-two per cent., the proteoses having correspondingly diminished ; but even at the end of seventeen days only fifty-four per cent, of pep- tones were to be found, thus affording striking evidence of the slow conversion of the first-formed products into pep- tones. Naturally, the individual proteoses show marked dififer- ences in their rate of conversion into secondary or final ' Chittenden and Hartwell : The Relative Formation of Proteoses and Peptones in Gastric Digestion. Journal of Physiol., vol. xii, p. 12. SOLUTION NOT STNOirTMOUS WITH PEPTONIZATION. 75 products. Take as an illustration some results' obtained with caseoses formed in the digestion of the casein of milk. Thus, heterocaseose, a primary product, yielded only fifteen per cent, of peptone after ninety-four hours at 40°C. with a strong pepsin-acid solution. Protocaseose, however, con- taining some deuterocaseose, under like conditions, yielded thirty-two per cent, of peptone in one hundred and nine- teen hours, while pure deuterocaseose gave sixty-six per cent, of peptone in one hundred and thirty-seven hours. Evidently, then, the first-formed soluble products of gastric digestion, *'. e., the primary proteoses, are only slowly con- verted into peptone, since they must first pass through the intermediate stage of deuteroproteose, which is plainly not a rapid process. The deutero-body, on the other hand, once formed is more rapidly converted into peptone, but even this is in no sense a rapid process. Hence, in the artificial digestion of proteids with pepsin-hydrochloric acid, solubility of the proteids may be quite rapid, and even complete in a very short time, but the resultant prod- ucts will be mainly proteoses and not peptones. The latter are truly formed and in considerable amount, but proteoses, either as primary or secondary bodies, are invari- ably present and usually in excess of the peptones. In this connection the question naturally arises how far we are to trust these results in their bearing on the natural process of digestion as it occurs in the living stomach. Obviously, the conditions are quite different in the two cases. In artificial digestions, we have especially the influence of an ever-increasing percentage of soluble prod- ucts on the activity of the ferment, a condition of things generally considered as more or less inhibitory to enzyme action. We have attempted to measure the real value of ' Chittenden and Hartwell, loc. cit., p. 23. 16 DIGESTIVE PEOTEOLTSIS. this influence by experiments' conducted in parchment dialyzing tubes, in which the conditions are made favorable for the removal of at least some of the products of diges- tion as fast as they are formed. In these experiments, the dialyzer tubes containing the proteid and pepsin-acid were immersed in a large volume of 0.2 per cent, hydrochloric acid (about three litres), which was gradually changed from time to time, the whole mixture being kept at 40° C. dur- ing the entire period of the experiment. The extent of peptonization was then ascertained by analysis of both the contents of the dialyzer tubes and of the surrounding acid, the results being compared with those obtained from con- trol experiments carried on in flasks. Without consider- ing the results in detail, it may be mentioned that the slow and incomplete peptonization so characteristic of artificial gastric digestion is not materially modified by this closer approach to the natural process. The several digestions carried on in the dialyzer tubes were certainly accompanied by a fairly rapid withdrawal of the diffusible products of digestion, yet no noticeable increase in the amount of pep- tone formed was observed. The results certainly favor the view that the conversion of the primary products of gastric digestion into true peptone is a slow and gradual process, even under the most favorable circumstances, and that this lack of complete peptonization is not due to accumulation of the products of digestion, but is rather an inherent quality of pepsin-proteolysis under all circum- stances. In these dialyzer experiments it was observed that not only did peptones diffuse, but also the proteoses. In fact, it was found that six to eight per cent, of the proteoses ^Chittenden and Amerman : A Comparison of Artificial and Natural Gastric Digestion, together with a Study of the DifEusibility of Prote- oses and Peptones. Journal of Physiol., vol. xiv, p. 483. DIFFgSIBILITT OF PEOTEOSES. 71 formed passed through the parchment walls of the dialyzer tubes into the surrounding acid in the nine hours' diges- tion. This led to a study of the difEusibility of proteoses in general, from which we were led to conclude that these bodies possess this power to a greater degree than had hitherto been supposed. As might be expected, it was also found that the attendant conditions modify materially the rate of di£Eusibility ; the two factors especially promi- nent being temperature and the volume of the surrounding fluid. Thus, 1.9 grammes of protoalbumose dissolved in 200 c.c. of water and suspended in 4.5 litres of water heated to 38° C, diffused through the parchment tube to the extent of 5.09 per cent., while at 10° C. diffusion amounted to only 2.5Y per cent. Under somewhat similar conditions, pure peptone diffused to the extent of eleven per cent, in six hours at 38° C. Somewhat singular, how- ever, was the result obtained with deuteroalbumose ; this proteose showing a diffusibility considerably less than that of the proto-body. But as Kiihne' has independently obtained essentially the same results, this apparent anomaly cannot depend upon any errors of work. It is of course to be understood that diffusion experi- ments made with dead parchment membranes cannot necessarily be expected to throw much light upon the rate of absorption of these bodies through the living membranes of the stomach and intestine, where, as Waymouth Reid ' has well said, we have to deal with an absorptive force dependent, no doubt, upon protoplasmic activity, and com- parable, in part at least, to the excretive force of a gland- cell. Furthermore, in considering absorption as it occurs in the living stomach, we must necessarily give due weight ' Erf ahrungen iiber Albumosen und Peptone. Zeitschr. f . Biol. , Band 39, p. 20. ' Osmosis Experiments ■with Living and Dead Membranes. Journal of Physiol., vol. xi, p. 312. 78 DIGESTIVE PKOTEOLTSIS. to the selective power of the epithelial cells, a power which may be far more potent even than we suppose. Hence, without attempting at this point to draw any broad deduc- tions from our experiments we may simply lay stress upon the facts themselves, viz., that the primary products of pepsin-proteolysis are diffusible, and, like true peptones, are capable of passing through animal and vegetable mem- branes, although to a less extent. We may further emphasize the fact that experiments of this character on diffusibility can, at the most, only indicate general tendencies, since every variation in the attendant conditions will exercise some influence upon the final result. With reference to the bearing digestive experiments made in dialyzer tubes have upon the natural process as carried on in the living stomach, we must necessarily grant that the conditions approximate only in the crudest way to those existent in the alimentary tract. At the same time, if complete peptonization is characteristic of pepsin-prote- olysis in the stomach, and failure to obtain such results in an artificial digestion is due to- lack of withdrawal of the diffusible products formed, then certainly the experiments carried on in dialyzer tubes, with abundant opportunity for diffusion, and with a large excess of free hydrochloric acid, should show some indications of increased peptone-forma- tion. But none were obtained. .It is more than probable that the rate of absorption of diffusible products from the stomach has been overesti- mated. Lea,' for example, assumes that, "normally the products of digestion, whether proteid or carbohydrate, are never met with in either the stomach or intestine in other than the smallest amounts, frequently to be described as merely traces." This certainly implies a far more rapid absorption of proteoses and peptones from the 'Journal of Physiol., vol. xi, p. 340. ABSOEPTION FROM THE STOMACH. 79 stomacli than results seem to justify. Indeed, recent facts obtained by Brandl,' working under Tappeiner's direction, tend to show that absorption from the stomach is, under some circumstances at least, comparatively slow. Brandl's experiments were conducted on large and vigorous dogs with gastric fistulse, the stomach being shut off from the intestine by the simple introduction of a small rubber balloon into the pylorus, which when dilated completely closed the orifice. By carefully conducted experiments, it was shown that pure peptone, entirely free from proteose, is absorbed from the empty stomach in proportion to the concentration of the peptone solution. Thus, 7.5 grammes of peptone dissolved in water in such proportion as to make a five per cent, solution, and allowed to remain in the stomach for two hours, lost by absorption only 0.28 gramme, equal to 2.68 per cent, of the peptone introduced. Under similar conditions, a ten per cent, aqueous solution of peptone lost only 4.6 per cent, by absorption. On the other hand, when peptone was introduced in larger quan- tity, viz., in a twenty per cent, solution, absorption amounted to thirteen per cent, in two hours. It is thus evident that pure peptones, even when taken into the stomach in fairly large amounts, and under con- ditions very favorable for rapid absorption, pass into the circulating blood very slowly. Obviously, however, one must not lose sight of the fact that when digestion is under way and the volume of blood consequently increased, there may be a corresponding rise in the rate of absorption. There is perhaps a hint of this conclusion in the influence of alcohol on the absorption of peptone as brought out by some of Brandl's experiments. Thus, it was found that when alcohol was added in considerable quantity to a ten ' Ueber Resorption und Secretion im Magen, imd deren Beeinflussung duroh Arzneimittel. Zeitschr. f. Biol., Band 39, p. 377. 80 DIGESTIVE PEOTEOLTSIS. per cent, solution of peptone, the stomach-nmcosa was greatly reddened, while in two hours the absorption of peptone amounted to 11.8 per cent. But in any event, these results certainly do not favor the view that the prod- ucts of gastric digestion are absorbed as soon as they are formed. It is, no doubt, quite different in the intestine, but in the stomach, where pepsin-proteolysis occurs, we have, I think, no grounds for assuming that either pep- tones or proteoses are rapidly absorbed. Hence, it might perhaps be considered that the results of pepsin-proteolysis in the living stomach are much the same as those obtained in artificial digestion experiments. Still, there are other differences between natural diges- tion and artificial proteolysis than those connected with the possible absorption of the more diffusible products of digestion. Thus, in the li^dng stomach there is an ever- increasing secretion of hydrochloric acid, and perhaps also of pepsin, more or less proportional to the extent of prote- olysis. On this point Brandl's experiments again give us some light. Thus, it was found that the introduction of an aqueous solution of peptone into the empty stomach led to the secretion of an acid fluid containing on an average 0.24: per cent. HCl, while, under similar conditions, the introduction of sugar or potassium iodide was followed by the secretion of a fluid containing on an average only 0.13 per cent. HCl. Further, the absolute amount of acid found after the introduction of peptone was far greater than when sugar or iodide was introduced, since peptone led to an increase of at least fifty per cent, in the volume of fluid secreted. Hence, proteolysis in the living stomach may give rise to such an increased production and secre- tion of hydrochloric acid that formation of the terminal products of gastric digestion may be greatly accelerated. That such in fact is the case, I have no manner of doubt, DIGESTION IN THE LIVING STOMACH. 81 but that it may result in the complete conversion of the so-called primary and secondary proteoses into peptone I very much question. In fact, such examinations as I have made of the stomach-contents after a suitable test- meal have always resulted in the finding of a relatively large amount of proteoses. To be sure, true peptone may be detected and in fairly large amounts, but whenever a quantitative determination of the relative proportion of the two has been made, the proteoses have always been in excess. I have already reported elsewhere the results of some experiments in this direction made on a healthy young man, where the stomach-contents were withdrawn at varying periods after the ingestion of weighed amounts of coagulated egg-albumin. Thus, in one experiment' the stomach was thoroughly rinsed with water, after which 138 grammes of finely divided coagulated-albumin, equal to 16 grammes of dry albumin, were ingested. Three-quarters of an hour thereafter, the stomach-contents were with- drawn by lavage and analyzed. As a result, 1.41 grammes of albumoses were separated and weighed, and 0.84 gramme of peptones, the relative proportion being expressed by sixty-two per cent, of albumoses and thirty-seven per cent, of peptones, calculated on the 2.25 grammes of soluble prod- ucts recovered. This expresses the general character of the results obtained in experiments of this nature, and in my opinion adds emphasis to the statement already made, that complete peptonization is not a feature of pepsin- proteolysis, either in the artificial or in the natural process as it takes place in the living stomach. Gastric digestion is to be considered rather as a prelimi- nary step in proteolysis, preparatory to the more profound changes characteristic of pancreatic digestion, in which the ferment trypsin is the important factor. We can thus see ' Journal of Physiology, vol. xiv., p. 501. 7 82 DIGESTIVE PROTEOLYSIS. how, as in the case of Czerny's dogs, an animal may he per- fectly nourished without a stomach, digestive proteolysis heing carried on solely by the pancreatic fluid. You will remember that two of the dogs operated on by Czerny and his pupils lived between four and five years after the operation, with the stomach completely removed, and yet during this period they were well nourished and ate all varieties of food with apparently a normal appetite.' Evidently, then, in some cases at least, digestive prote- olysis can be carried on without this preliminary action of the gastric juice. Ogata" arrived at essentially the same conclusion by the establishment of a duodenal fistula, shut- ting off the stomach from the intestine by means of a small rubber ball which could be inflated with water. On then introducing coagulated egg-albumia and other forms of proteid matter into the duodenum, he found that digestion was at least suflSciently complete to satisfy all the demands >of the system. The only unsatisfactory result was with I collagenous foods, which plainly showed the need of a pre- I liminary acid digestion. More recently still, Cawallo and ^Pachon,' working in Richet's laboratory, have studied the digestibility of different kinds of proteid foods in a dog, upon which they had performed a gastrectomy; the entire fundus, as well as the pyloric portion, of the stomach having been removed. In an animal so operated upon, after recovery was complete, solid food, as meat, was com- pletely digested when taken in small quantities at a time. Haw meat, however, was less completely utilized, the faeces showing portions of undigested fibres. Still, it was apparent that intestinal digestion alone was capable of ' Bunge's Physiologisohe und Pathologische Chemie, p. 153. ' Ueber die Verdauung naoh der Aussolialtniig des Magens. Du Bois- Eeymond'B Arohiv f . Physiol, 1883, p. 89. ' Une observation de chien sans estomao. Comptes Eendus bebd. de la Soci^W de Biologie, December 1, 1893. DIGESTION WITHOUT A STOMACH. 83 accomplishing all that was necessary for the complete nourishment of the animal, when it had once become accustomed to the changed condition of its alimentary tract. These facts are cited not to belittle the importance of gastric digestion in the nutrition of the body, but rather to emphasize the probability that pepsin-proteolysis is simply a preliminary step in digestion ; that its function is not in the direction of a complete peptonization of the proteid foods ingested, but that its action is especially directed to the production of soluble products, proteoses, which can be further digested in the small intestine, or perhaps directly absorbed after they have passed through the pylorus, or even from the stomach itself to a certain extent. SOME PHYSIOLOGICAL PROPERTIES OF PROTEOSES AND PEPTONES. It is very evident from what has been said that all forms of proteid matter, i.e., all the members of the three main groups spoken of in our classification of the proteids, excepting only nuclein, recticulin, and the keratins, are capable of undergoing proteolysis with pepsin-hydrochloric acid. Further, in every case the main products of the transformation are proteoses ; viz., albumoses, caseoses, gelatoses, vitelloses, myosinoses, etc., according to the nature of the proteid undergoing proteolysis ; true pep- tones being formed in less abundance. Corresponding to each of these groups are primary and secondary proteoses, all possessed of many points in common, both chemical and physiological, yet differing from each other in many minor respects. These are the important products of gastric digestion, of pepsin-proteolysis, and it may be well 84 DIGESTIVE PROTEOLYSIS. to consider for a moment some of the physiological proper- ties of the proteoses and of peptones as well, in order that we may the better comprehend the general nature of these substances with reference to their possible action in the economy. , As far back as 1880, Schmidt-Mulheim' discovered that the injection of aqueous solutions of peptone into the blood-vessels of living dogs was attended by a series of remarkable phenomena. Thus, the animal passed at once \ into a condition of narcosis resembling that produced by chloroform, accompanied by a fall of general blood- pressure so great that the animal was liable to die, as from asphyxia. Further, there was evidence of some marked change in the condition of the blood, as indicated by loss of the power of spontaneous coagulation, while the peptone itself evidently underwent some alteration, or else was rapidly eliminated, since it could not be detected in the blood a short time after its introduction. These experi- ments, however, were not conducted with true peptone but with "Witte's " peptonum siecum," which at that time, at least, was composed in great part of proteoses. The general character of these interesting results was confirmed by Fano," who found that the injection of so-called pep- tone in the proportion of 0.3 gramme per kilo, of body- weight was suflHeient to bring about complete narcosis, together with loss of coagulability on the part of the blood. Very suggestive, however, was the fact that Fano, on try- ing similar experiments with the peptone formed by pan- creatic digestion, viz., with antipeptone, which presumably contained a far smaller proportion of proteoses, failed to obtain like results ; the tryptone, so-called, being exceed- ' BeitrSge zur Keuntniss des Peptous uud seiner physiologisohen Bedeutung. Du Bois-Eeymond's Archiv f. Physiol., 1880, p 33. ' Das Verhalten des Peptons und Tryptons gegen Blut und Lymphe. Ibid., 1881, p. 377. PHYSIOLOGICAL ACTION OF ALBUMOSES. 85 ingly irregular in its action, in many cases producing no effect whatever. The discovery at this date of the several albumoses, and their presence in large amounts in all so-called peptones, led to a study of their physiological action with special reference to the observations of Schmidt-Miilheim and , Fano. Politzer," working under Kiihne's guidance, was the first to experiment in this direction, and his results are full of interest as throwing light on the action of the individual albumoses. Thus proto, hetero, and deutero- albumose are all active physiologically, giving rise when injected into the veins of dogs and cats to strong narcotic action, varying somewhat in intensity in different individ- uals. There is also produced a marked fall in blood- pressure, due apparently to vaso-motor paralysis, the action being manifested chiefly, if not wholly, on the splanchnic region. Thus, after an injection of one of these albumoses, the mesenteric vessels are always strongly congested, accompanied frequently by the appearance of a bloody serum in the peritoneal cavity. Narcotic action is mani- fested only so long as the blood-pressure remains sub-nor- mal, and is due presumably to this marked accumulation of blood in the large abdominal veins, thus leading to anaemia of the brain. Albumoses and peptones injected into the jugular vein likewise produce fever, presumably through some action on the nervous system by which the equilib- rium of tissue-metamorphosis is interfered with.° Further, Politzer found that all of the albumoses either delayed or prevented altogether the coagulation of the blood, in conformity with the observations of Schmidt- Miilheim and Fano. In all of these actions the primary ' On the Physiological Action of Peptones and Albumoses. Joiinial of Physiol., vol. 7, p. 383. ' Ott and CoUmar : Pyrexial Agents, Albumose, Peptone, and Neurin. Journal of Physiol., vol. 8, p. 218. 86 DIGESTIVE PROTEOLYSIS. albiimoses appeared most effective, deuteroalbumose least so. Heteroalbumose, however, was constantly most active, especially in delaying the coagulation of the blood. "With amphopeptone, there was far less narcosis and less diminu- tion of blood-pressure, while the effect on the coagulability of the blood was more or less variable, frequently being entirely negative. Antipeptone, on the other hand, was found almost wholly wanting in any constant effects, although in one instance deep narcosis was produced. Thus, from Politzer's experiments, it was made clear that the albumoses, when introduced directly into the blood- current, possess a far greater toxic action than either amphopeptone or antipeptone. Albumoses, in sufficiently large doses, were invariably fatal, while peptones never produced fatal results so long as the kidneys of the animal remained intact. The extreme solubility and diffusibility of peptones, coupled perhaps with their marked diuretic action, lead to rapid elimination through the kidneys, and their consequent removal from the system. Many of these observations made with the albumoses I have repeated with several of the proteoses and peptones more recently studied, as protocaseose, protoelastose, the globuloses, and others. The results may be taken as prac- tically confirmatory of the older observations, and I make mention of them in this general way simply to emphasize the fact that all of the proteoses, though perhaps showing individual peculiarities, are possessed of marked physiologi- cal properties, which plainly testify to their toxic nature, when introduced directly into the blood-current. Young animals are particularly sensitive to the injection of proteoses into the blood, even when the introduction takes place very gradually.' Thus, a young, healtliy dog ' Neumeister : Ueber die Einf iihrung der Albumosen und Peptone in den Organismus. Zeitsohr, f. Biol., Band 24, p. 284. INTEODtrCTION OF PROTEOSES INTO THE BLOOD. 87 of 2 kilos, body-weight, eight weeks old, died in one hour after the injection into the jugular vein of 1 gramme of protoalbumose in 20 c.c. of water, thus affording a good illustration of the extreme toxicity of this albumose when introduced directly into the blood. Of greater interest, physiologically, are the changes the individual proteoses undergo after their injection into the blood. As already stated, peptone so injected may appear in the urine wholly unaltered. Thus, Neumeister' has made injections of both amphopeptone and antipeptone in the case of dogs, and was able to detect the peptone very quickly in the urine. I have made like experiments with other forms of peptone and obtained similar results ; thus, a pure amphopeptone formed from casein by pepsin-prote- olysis (2 grammes in 15 c.c. water) was injected into the jugular vein of a dog weighing 5 kilos. The urine col- lected during several hours after the injection was heated to boiling, and saturated while hot with ammonium sulphate. The filtrate, on being tested with cupric sulphate and potassium hydroxide, gave a fairly strong biuret reaction for peptone. Another similar experiment made with antipeptone, formed from the myosin of muscle-tissue, gave like results. With proteoses, however, different results are obtained, as JSTeumeister" 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 kid- ' Zeitsohr. f. Biol., Band 34, p. 387. = Ibid., p. 384. 00 DIGESTIVE PEOTBOLTSIS. neys 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 knowl- edge. 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 kid- neys 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 Stadelinann' 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. However this may be, it is very evident that the pro- teoses 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 con- clusion that they are truly foreign substances, and the sys- tem 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 pep- sin-proteolysis undergo some transforination 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, how- ever, will be left until the next lecture. ' Untersuohungen fiber den Pepsin-fermentgehalt des normalen und pathologisohen Hames. Zeitschr. f. Biol., Band 35, p. 308. POISONS PRODUCED BY BACTERIA. 89 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 bacte- ria are proteose-like bodies, chemically, at least, closely akin to the proteoses resulting from pepsin-proteolysis. Thus, Wooldridge' 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 intro- duced into the blood it possessed the power of producing immunity to anthrax. This observation was verified by Hankin," 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^ carried the matter still further, and by growing the anthrax-bacilli in a pure solution of alkali-albuminate pre- pared 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 pro- nounced 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 com- posed 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. Albumose-like bodies have also been obtained by Brieger ' Versuche fiber Schutzimpfung auf ohemisehem Wege. Dn Bois-Eey- mond's Arohiv f. Physiol., 1888, p. 527. i* British Med. Journal, October, 1889. » Proceed. Royal Society, 1890, vol. 48, p. 78. 90 DIGESTIVE PEOTEOLTSIS. and Franker 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 resembliug those produced by the Loffler 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, albumo- ses, peptones, and crystalline bodies such as leucin and tyro- sin are formed in considerable quantities. Kresling" has reported similar results. With the tubercle-bacilli, many like results have been recorded. Thus, among others, Crookshank and Herroun^ 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. Koch* 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' to have a somewhat peculiar composition, inas- much 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.' Still more recently, Kiihne' 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 acrooalhu- moses. They are endowed with marked chemical and ' Untersuoliuiigeii iiber Bacteriengifte. Berlin, klin. Woohenschrift, 1890, p. 341 and 268. ' Ueber die Bereitimg des Malleins Tind seine Bestandtheile. Abstract in Jahresbericbt f . Thierehemie, Band 22, p. 634. * Journal of Physiology, vol. 13, p. 9. * Deutsche med. Woohenscrift, 1891, p. 1180. = lUA. ' Chittenden : On the Proteolytic Action of Bromelin, the Ferment of Pineapple Juice. Journal of Physiology, vol. 13, p. 303. ■■ Weitere Untersuohungen iiber die Proteins des Tuberculins. Zeit- sohr. f. Biol., Band 30, p. 321. TOXTC NATTTEE OF PEOTEOSBS. 91 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. 1 wish merely to call attention to the fact that the pro- teoses, and likewise the peptones formed by pepsin-prote- olysis, are more or less toxic when introduced directly into the blood, and that they share this property with the prote- oses 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-prote- olysis must undergo some transformation, before reaching the blood-current, by which their peculiar physiological properties are modified. This modification may be asso- ciated with a conversion into the serum-albumin, or globu- lin 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 92 DIGESTIVE PEOTEOLTSIS. body, but between their formation as a result of proteolysis and tbeir passage into the blood they are exposed to some agency, or agencies, doubtless in the rery 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. LECTUKE III. PROTEOLYSIS BY TEYPSIN— ABSORPTION OF THE MAIN PEODUCTS 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 conse- quently 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 testi- fies 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 pro- duction of soluble proteoses and peptones, but the hemi- portion of the latter is quickly broken down into crystalline bodies, such as leucin, tyrosin, lysia, lysatin, etc. This special characteristic of the ferment testifies in no uncer- tain manner to the existence of inherent qualities in the 94 DIGESTIVE PROTEOLYSIS. 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 produc- tion of proteoses and peptones, while trypsin gives rise to a' series of hydrolytic cleavages which result iu 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- reactiug 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, iu experiments' on blood-fibrin it was found that, while a solution of trypsin containing 0.5 per cent, sodium carbon- ate, 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 condi- tions, 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. "With hydrochloric acid, trypsin is quickly destroyed, unless there is a large excess of proteid matter present," ' Chittenden and Cummins: Studies in Physiol. Chem., Yale Uni- versity, vol. i., p. 135. ' Mays : TJntersuohungen aus d. pliysiol. Institute d. ITniversitSt Heidelberg, Band iii., p. 378 ; also Langley : On the Destruction of Fer- ments in the Alimentary Canal, Journal of Physiology, vol, iii., p. 263. TRYPSIN A PEPTONE-FOEMING FEEMENT. 95 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, espe- cially 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 vary- ing circumstances, frequently prevail in the small intestine. 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 con- version 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 deutero- proteoses,' which are then transformed by the further ' E. Neumeister : zur Kenntniss der Albumosen. Zeitsclir. f. Biol. Band 33, p. 378. 96 DIGESTIVE PEOTEOLTSIS. 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. Ampliodeuteroproteoses. Amphopeptones. Antipeptoue. Hemipeptone. Leucin. Tyrosin. Aspartic acid, etc. 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 T5° C.,' 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 fiuid 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. The fact that deuteroproteoses are the primary products of trypsin-digestion again emphasizes the natural adaptar bility 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 ' Jao. G. Otto : BeitrSge zur Kenntniss der Umwandlung von Eiweiss- BtofEen durch Pancreas-ferment. Zeitschrift f. physiol. Chem., Band 8, p. 129. FATE OF HEMI-GEOUPS IN TEYPSIN-PEOTEOLTSIS. 97 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 deutero- proteoses, peptones, and crystalline amido-acids. It is to he remembered, however, that in trypsin-proteolysis the deuteroproteoses and the amphopeptones must necessarily be represented by bodies in which there is a preponder- ance 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 hemipep- tone formed from any native proteid can be converted into crystalline and other products, thus leaving a true anti- peptone 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 pan- creatic digestion. Still again, it is to be remembered that in normal digestive proteolysis, as it occurs in the living 98 DIGESTIVE PROTEOLYSIS. 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 prod- ucts 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-albumiu 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 prod- ucts, 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 tryp- sin, 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 sim- ple products, while the anti-portions remain unchanged, being resistant to the ferment and thus leading to a sepa- ration of the two groups, or at least to the isolation of the anti-molecules. ANTIPEPTONES. 99 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 pancre- atic 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 foimd a small amount of a peculiar antialbumid- like body, insoluble in the weak alkaline fluid. Antipep- tones, 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 blood-flbrln.i From blooa-llbrln.2 From From antlalbomose.s casein.* From myosin. B 47.30 49.59 48.94 49.94 49.36 H 6.73 6.93 6.65 6.51 6.87 N 16.83 15.79 15.89 16.30 16.63 S 0.73 — — 0.68 1.16 38.41 — — 36.57 36.09 Eul me and Chittenden : Studies in Physiol. Chem. Tale TJniver. vol. ii., p. 40. * J. Otto : Zeitsohr. f. physiol. Chem., Band 8, p. 146. ' Kuhne and Chittenden: Zeitsohr. f. Biol., Band 19, p. 196. * Chittenden : Studies in Physiol. Chem. Yale Univer. , vol. iii., p. 101, ^ Chittenden and Goodwin : Journal of Physiol., vol. xii., p. 34. 100 DIGESTIVE PEOTEOLYSIS. 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 correspond- ingly high content of carbon and a low content of nitro- gen. 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 5Y.48 per cent, of carbon, Y.6T per cent, of hydro- gen, 13.94 per cent, of nitrogen, 1.32 per cent, of sulphur, and 19.59 per cent, of oxygen.' It is only necessary to compare tliese figures with those expressive of the compo- sition 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, how- ever, is a peculiar product, one which is liable to crop out somewhat unexpectedly, and with varying shades of resist- ance 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 insolu- ble product may be quite large, even reaching to one- fourth of the total proteid matter ; '' 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 ' Chittenden and Goodwin : Journal of Physiol., vol. lii., p. 36. ' KUhne und Chittenden ; Ueber die nSehsten Spaltungsproduote der EiwelsskSrper. Zeitsohr. f. Biol., Band 19, p. 196. FOEMATION OF AMIDO-ACIDS, ETC. 101 mixture, it may be dissolved in dilute caustic alkali, repre- cipitated 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 anti- albumid is prone to separate out as a gelatinous coagulum, more or less resistant to the further action of the ferment. 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 fer- ment 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 ten- dencies 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 con- sidered as amido-isobutylacetic acid, (CHj);, CH CH, CH (NHj) COOH ; and tyrosin, a body belonging to the aromatic OTT group, having the formula C,H,