with in each case the addition of 0-2 0/ acetic acid. The methods added to the juice in part of calcium oxalate formed from the oxalate

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ON THE PRODUCTS OF THE PROTEOLYTIC ACTION OF AN ENZYME CONTAINED IN THE CELLS OF THE SPLEEN. BY J. B. LEATHES, Department of Pathological Chemistry, Jenner Institute, London.

1 ON THE PRODUCTS OF THE PROTEOLYTIC ACTION OF AN ENZYME CONTAINED IN THE CELLS OF THE SPLEEN. BY J. B. LEATHES, Department of Pathological Chemistry, Jenner Institute, London. THAT the spleen cells contain a proteolytic enzyme, which acts far more powerfully in an acid medium (0 2 0/0 acetic acid) than either in a neutral or alkaline one, was recently shown by Hedin and Rowland(l). The method employed by themii to determine the degree of activity is fully described in their paper, and consists in the determination of the nitrogen in the filtrate from the precipitate given with tannic acid; the stronger the action the more nitrogen escapes precipitation by this reagent. A similar if not identical enzyme was shown by them subsequently to be present in the cells of other organs, lymphatic glands, kidneys, liver, heart muscle: while in the voluntary muscles the enzyme found acted equally in acid, netitral, and alkaline fluids, but was comparatively inactive in all (2). In order to determine what the nature of the nitrogenous products of the enzyme action are, I have investigated in the first place the solution obtained from the action of filtered ox spleen juice, prepared as described by Rowland (8), upon large quantities of fibrin, and secondly also the fluid juice after it had been kept for some weeks at the body temperature under a layer of toluol without the addition of fibrin, but with in each case the addition of 0-2 0/ acetic acid. The methods adopted for the separation of the various substances ini solution in the fluid were in the main those adopted in similar investigations by Kossel and Kutscher (4). The digestion was allowed to proceed for seven weeks in two cases, and for from three to four months in two others. The resulting fluid always contains a certain amount of insoluble matter, which consists in part of substances of the nature of nucleins, in part of haematin, in part of tyrosin crystals, and where fibrin had been added to the juice in part of calcium oxalate formed from the oxalate used in collecting the blood. This residue was filtered off. The filtered acid fluid containing all the soluble products of digestion

2 ENZYME OF SPLEEN. 361 gives on boiling a coaguluim, which is increased in amount by the addition of further quantities of acetic acid, and is not all removed till the amount of acetic acid reaches at least 05 0/0. This coagulum was filtered off. After removal of the coagulum the fluid still gives the biuret reaction, though only faintly. On testing for albumoses, by fractional precipitation with ammonium sulphate, it was always possible to show their presence, though usually only in the merest traces. In three samiples digested for from seven to eight weeks, it was made out that the fraction coming down on half saturation, and consisting therefore of proto- and hetero-albumose, was considerably in excess of any later fraction consisting of deutei o-albumoses. In the first fraction there was a definite thouigh small precipitate, in the later fractions a faint turbidity, which on standing for 24 hours amounted only to a few filmy flakes. These traces of albumoses were therefore neglected. The fluid was then treated with bariumi hydrate in the cold so long as a precipitate was formed, filtered, and any excess of barium having been removed with sulphuric acid, concentrated to about a tenth of its oriainal volume on the water-bath. OIn standing in the cold for a day the fluid turned to a thick paste of crystals, in which the forms characteristic for impure leucin preponderated, though tyrosin sheaves were also abundant. These were filtered off on the pump and the fluid fractionally precipitated with silver nitrate, as done by Kossel and Kutscher. The first fraction, obtained by adding silver nitrate to the fluid made faintly acid to congo with nitric acid, should have contained the greater part of the xanthin bases present. The precipitate was however soluible in amnmonia, and I may here say that though I have not yet systematically searched for xanthin bases, I have not as yet come across them where they might have been expected to be present. A feature of the method of preparing the juice is that certain organic substances seem to be selectively retained in the press-cake, though whether, as is possible, the xanthin bases are among them I cannot say. This first silver precipitate, after removing the silver with sulphuretted hydrogen, was treated with phosphotunigstic acid, and the precipitate obtained on liberation from the acid gave the biuret reaction, but could not be separated in any analysable form. The fluid filtered from the acid silver precipitate was treated with more silver nitrate and then with saturated cold barium hydrate solution so long as a filtered sample gave a precipitate with weak

$The solution thus obtained was separated into a basic and an acid fraction by means of phosphotungstic acid and 5 0/0 sulphuric acid.$

3 362 J. B. LEA THES. ammonia, and the abundant bulky precipitate was filtered off, washed, and decomposed with sulphuretted hydrogen in the presenice of sulphuric acid. The solution thus obtained was separated into a basic and an acid fraction by means of phosphotungstic acid and 5 0/0 sulphuric acid. The basic fractioni, freed from these reagents by baryta, was precipitated again with silver and ammonia. The precipitate, which was entirely soluble in ammonia and therefore contained no xanthin bases, was washed and decomposed with hydrochloric acid. The acid fluid on concentration gave crystals, which after decolorization and recrystallization were dried gr. gave AgCl= Cl= /o, Calculated for histidin bichloride Cl =31-140/o The acid fraction, not precipitated by phosphottungstic acid, was freed exactly from the inorganic acids andtreated with copper carbonate on the water-bath for some hours, filtered hot, and on cooling gave an abundant crystallization of a copper salt having the appearance of the salt of aspartic acid. Recrystallized and dried in vacuo at 1000 C. the crystals were found to contain /0 nitrogen: the anhydrous copper salt of aspartic acid requiring /o. The filtrate from the second silver precipitate gave a third group of silver compounds on addition of more silver and saturation with barium hydrate. These were washed with baryta water and decomposed with sulphuretted hydrogen and sulphuric acid, the bases contained in them separated by phosphotungstic acid, set free by baryta and after exact removal of the baryta and concentration, treated with silver nitrate. A precipitate of carbonate of silver was removed and the fluid concentrated in the presence of calciumn carbonate in order to separate the comparatively insoluble basic compound of arginin and silver nitrate. On cooling, crystals were obtained which were recrystallized several times, and the silver that they contained estimated as chloride gr. gave Ag = /0, Calculated for C6H14N402. AgNO3 + JH2O. Ag = /0. After removal of the insoluble silver compounds, silver and barium were separated from the filtrate as stilphide and sulphate and the fluid treated with phosphotungstic acid. The precipitate decomposed with baryta, and freed from excess by carbonic acid, gave on concentrating the solution obtained, a smell suggesting the presence of diamines: but in the picrates obtained, beyond lysin and potassium, no other bases could be isolated either by the method of Lawrow (5), or on benzoylation. The

ENZYME OF SPLEEN. picrate of lysin was converted into the bichloride and crystallized from alcohol. 0-2664 gr. gave 0,3471 AgCl = 0-0858 C1= 32-23 0/0, Calculated for C0H1,N202. 2HC1 = 32 42 0/0.

4 ENZYME OF SPLEEN. picrate of lysin was converted into the bichloride and crystallized from alcohol gr. gave 0,3471 AgCl = C1= /0, Calculated for C0H1,N202. 2HC1 = /0. In another experiment a modification of the above procedure was adopted; the fluid, after filtering and removing coagulable substances, was concentrated and freed from the substances that crystallized out, and then at once treated with phosphotungstic and sulphuric acids. The basic portion thus obtained yielded histidin, arginin and lysin, which were separated as before by the use of silver; the acids in the filtrate were investigated separately. A preliminary separation by mercuric chloride and baryta was effected, the object of wbich was to remove aspartic acid, and if present glutainic acid. Special experiments showed that pure aspartic acid was almost completely thrown down in this way (97 0/o) and glutamic acid nearly as completely (91 0/o). The filtrate was then divided into two portions: one-half was treated with copper carbonate, filtered, evaporated to dryness and extracted with alcohol, and the copper salts soluble in alcohol separated from those insoluble in alcohol; the other half was after concentration treated by Emil Fischer's method with alcohol, and saturated with hydrochloric acid. The esters obtained from the latter were distilled in vacuo, in fractions coming over between 35C and 650, 650 and 850, 8a50 and 1000, the second being much the most abuindant. This fraction, saponified and concentrated, gave crystals which were fractionated. The first fraction (3-1 gr.) recrystallized from water gave a crop of glistening white scales with a soapy feeling, amounting to 07 gr.: of these gr. by Kjeldahl's method gave NH= 22 c.c. N H2SO4, N = = /0. Leucin contains N =10-69 /o The third fraction (1-2 gr.) recrystallized gave crystals of a different form, rosettes of small plates, much harder than those obtained from the first fraction, and not soapy to touch, nor scaly. N 0'2482 gr. by Kjeldahl's method gave NH.3= 21 c.c. 10 H2SO4, N = = 11P84 0/0. Amido-valerianic acid contains N = /o The mother-liquor fromn which these three fractions of crystals were obtained was treated with copper carbonate, evaporated to dryness and extracted with absolute alcohol. But neither fronm these copper salts, nor N 363

5 364 J. B. LEATHES. from the copper salts obtained from that half of the acids not precipitated by mercury which was not converted into esters, could I obtain any satisfactory crystalline preparation of the other amido acids isolated from this fraction of the esters by Emil Fischer, the active and the inactive pyrrolidin carbonic acids, although substances were present in each case which behaved as these acids would. Probably the multiplicity of the processes to which they had been submitted would have affected them, and it is likely that by a more direct search I may find them. To sum up therefore the cleavage products that have been identified as being formed are leucin, tyrosin, amido-valerianic and aspartic acids, arginin, histidin, and lysin. In addition to these haematin, identified spectroscopically, is present in large amount, in the insoluble residue, and to a certain extent too in solution. The digested fluid gave in every case the tryptophan reaction very strongly in the first instance: the prolonged and varied treatment, however, which was necessary for the isolation of the substances not characterized by any single definite reaction, seems to have altered this body and the residual fluids no longer gave this reaction. A special experiment with fresh material will be necessary to prove the identity of the substance in digested spleen juice with the substance tryptophan isolated by Hopkins and Cole (6). The fact that the digested juice, at a period when albumoses are present in it only in traces, still contains comparatively large quantities of coagulable proteids, is remarkable. With regard to the coagulum the following points were ascertained: it dissolves readily in weak caustic alkalis, and even in alkaline carbonates: from the solutions it is reprecipitated by acids: after purification by repeating this process three or four times the substance contains phosphorus as well as sulphur: it gives the proteid reactions, biuret, Millon an(1 glyoxylic. The question whether glutamic acid is formed I am not at present able to answer. The methods for isolating glutanmic acid are perhaps not very delicate. But neither by the original methods of Habermann and Hlasiwetz (7), nor by that suggested by Kutscher (8) could I get any positive proof of the presence of this acid. The same applies to cystin: among the crystals to be seen in the insoluble products of the action of the ferment sometimes there are some that suggest the presence of cystin. But an ammoniacal extract of this residue has always failed to give evidence of this when tested with lead and cauistic soda. The separated crystals have always readily dissolved in nitric acid (9), and any material insoluble in this reagent,

6 ENZYME OF SPLEEN..365 dissolved in soda, also failed to give this reaction. An unstable sulphur compound, which gives up its sulphur as hydrogen sulphide to hot alkalis, anid is soluble in ether, is present, but I am not yet.in a position to define its nature and properties more exactly. CONCLTJSIONS. The products of the actiotn in an acid medium of the proteolytic enzyme found in cell juice from the spleen of oxen are the same as those found to be formed by trypsin in alkaline media or those formed by the hydrolytic action of mineral acids. As to the physiological significance of such an enzyme, should those found in the cells of other organs be similar, as is probable, the difficulty that the reaction most favourable to its action is an acid one cannot be said to exclude the possibility of such significance till more is known of the balance of molecular forces within the cell. There seem for instance to be grounds for the view suggested by Ehrlich that the reaction of the nucleus may be acid (1O). Granted, however, the conditions favourable for its action, the fact that the products formed are identical with those formed in the extracellular digestion of proteid foods, suggests that intracellular proteid catabolism is essentially a reversion of what we must infer, from our knowledge of the digestion of foods, to be the course of intracellular proteid anabolism. The same fact possibly throws light on the familiar phenomenon of starvation, where the autolysis of proteid in muscles and certain viscera, the spleen included, provides the heart and nervous system with identical substitutes for the products of the digestion of proteid foods. REFERENCES. (1) Hedin and Rowland. Zeitschrift fur physiol. Chemie, xxxii (2) Hedin and Rowland. Ib. p (3) Rowland. Journal of Physiology, xxvii. p (4) Cf. especially Kutscher, Zeitschrift fur physiol. Chemie, xxxii. p (5) Lawrow. Zeitschrift fur physiol. Chemie, xxxiii. p (6) Hopkins and Cole. Journal of Physiology, xxvii. p (7) Habermann and Hlasiwetz. Liebig's Annalen, clxix. p (8) Kutscher. Zeitschrift fur physiol. Chemie, xxxii. p (9) Embden. lb. p. 94. (10) Ehrlich and Lazarus. Die Anieiuie, Pt. i. p. 31, etc. 23-5

investigated later by Hed in(3). It was accordingly of some interest to

investigated later by Hed in(3). It was accordingly of some interest to ON THE PRODUCTS OF DIGESTION OF THE PRO- TEOLYTIC SPLEEN ENZYME ACTING IN AN ALKALINE MEDIUM. BY E. P. CATHCART, M.D., Research Student in Pathological Chemistry, Lister Institute, London. SOME two years