PAPOID-DIGESTION. BY V R. H. CHITTENDEN, Ph.D. Professor of Physiological Chemistry in Yale University. PAPOID-DIGESTION. [From the Transactions of the Connecticut Academy, Vol. TX, 1892.] Papoid-digestion. By R. H. Chittenden. “ Papoid,” a therapeutic agent, noted especially for its peculiar proteolytic power, is prepared from various parts of the papaw plant, Carica Papaya. As pointed out by Martin,* the occurrence of such a proteolytic ferment in the vegetable kingdom is in itself remarkable, and its discovery, together with that of other vegetable ferments, may eventually throw important light on the assimilation of animal food by carnivorous plants, as well as on the character of the proteolytic changes in the reserve proteids of plants in general. Furthermore, such vegetable ferments when properly isolated may prove of great value in medicine as therapeutic agents, and it is with this point in view that the following study of papoid has been undertaken. The researches of Martinf have shown that papaw juice is pecu- liarly rich in a variety of proteids, with one or more of which the proteolytic ferment is associated. Hence, it would naturally be ex- pected that any active preparation of this ferment would contain a large proportion of proteid or albuminous material. This is the case with papoid, and the proteids present, as seen from the follow- ing reactions, are of several kinds. 1. General Reactions of Papoid. Papoid, treated with distilled water, yields on filtration, a yellow- ish colored solution, leaving a small, fiocky, insoluble residue. The solution is almost neutral, showing, however, a faint alkaline reac- tion when tested carefully. A drop or two of dilute acid does not, however, give any neutralization precipitate. The matter insoluble in water is partially dissolved by a 5 per cent, solution of sodium chloride, the fluid giving a fairly heavy precipitate with concen- trated nitric acid, which on heating turns yellow, but does not dis- solve, thus indicating the presence of a globulin. This residue of globulin is also soluble in 0 5 per cent, sodium carbonate and in 0‘2 per cent, hydrochloric acid, from both of which solutions it is re- precipitated by neutralization, again dissolving in a slight excess of either dilute acid or alkali. That portion of the residue not dis- * Journal of Physiology, vol. 5, p. 213. -f- Ibid., vol. 6, p. 341. It. H. Chittenden—Papoid-digestion. 3 solved by salt solution, dilute acid or alkali, is composed mainly of insoluble or coagulated proteid. The addition of distilled water to a clear, aqueous solution of papoid produces a pronounced turbidity, which disappears at once on the addition of a little salt solution, thus showing the presence of a globulin which is obviously held in solution by virtue of the salts contained in the preparation. Concentrated hydrochloric acid added to a clear, aqueous solution of papoid gives a heavy white precipitate of proteid matter, readily soluble in excess of the strong acid. Concentrated nitric acid, under similar circumstances, produces a heavy white precipitate, which on heating or boiling dissolves in great part, yielding a reddish-yellow solution. On cooling this solution, the precipitate reappears, dissolving again as the mixture is heated, and once more reappearing as the tiuid cools. This reac- tion is due to the presence of an albumose, while the accompanying body precipitated by nitric acid, but insoluble on heating, is a globulin, presumably the same as that previously noted in the in- soluble matter. Acetic acid and potassium ferrocvanide produce a heavy white precipitate only in part dissolved by warming. Boiling a clear, aqueous solution of papoid gives rise to a tur- bidity, which on prolonged boiling passes into a flocculent precipi- tate. This precipitate is insoluble in 0-2 per cent, hydrochloric acid, and is therefore presumably composed of coagulated globulin. Addition of magnesium sulphate in substance to an aqueous solu- tion of papoid precipitates the globulin present, with perhaps some albumose. At first sight, from the milky appearance of the fluid, the precipitate appears quite voluminous, but the amount is in reality not large. On adding crystals of sodium sulphate to the filtrate from the above precipitate, in such quantity as to insure complete saturation of the fluid and with formation of sodio- magnesiuin sulphate, a second heavier precipitate is produced, com- posed mainly of albuinoses (one or more) which agglutinates into a somewhat gummy mass, especially on the addition of a drop or two of acetic acid. With this precipitate of albumose, the proteolytic ferment appears to be mainly associated. In the filtrate from this second or sodio-magnesium sulphate pre- cipitate, the presence of peptone, in considerable quantity, can be shown by the biuret test. Or, by directly saturating an aqueous solution of papoid with ammonium sulphate while warm, the albu- 4 R. H. Chittenden—Papoid-digestion. moses and globulin can be precipitated together, while the peptone can be detected in the filtrate by the biuret test, viz: by potassium hydroxide* and dilute cupric sulphate solution. From the foregoing simple reactions, it is evident that papoid is composed essentially of a mixture of globulin, albumoses and pep- tone, with which is associated the ferments characteristic of the preparation. This is essentially in accord with what is known re- garding the vegetable ferments in general, and indeed, the animal ferments as well. Thus even pepsin, in its general chemical reac- tions, behaves like an albumose, and the best known methods of isolating vegetable ferments result simply in the separation of one or more albumoses, or a glodulin-like albumose, with which the fer- ment appears to be inseparably connected. In addition to the above proteid constituents, papoid appears to contain a small amount of indifferent material, probably added to counteract any tendency which the peptones or other like bodies have towards the accumula- tion of hygroscopic moisture. 2. The Proteolytic Action of Papoid. As early as 1874, Royf had called attention to the fact that papaw juice had the power of dissolving both animal and vegetable albumin, although he apparently did not clearly recognize the pro- cess as one of digestion. Later, AlbrechtJ experimented in the same direction, and since then many experimenters have added their testimony to the power of papaw preparations as solvents for pro- teid matter. Papoid, so far as my observations extend, has the power of digest- ing to a greater or less extent all forms of proteid or albuminous matter, both coagulated and uncoagulated. Furthermore, papoid is peculiar in that its digestive power is exercised in a neutral, acid and alkaline medium. These statements are amply illustrated by the following experiments : a. Action on Coagulated Egg-albumin. The albumin was prepared for this experiment by taking the whites of several eggs, cutting the transparent membranes with scissors, adding an equal volume of distilled water and straining the mixture through fine muslin to remove the meshes enclosing the * The potassium hydroxide solution must be added in large excess, sufficient to decompose all of the ammonium salt present. f Glasgow Med. Journal, 1874. J Schmidt’s Jahrbuch, vol. 190. R. II. Chittenden—Papoia-digestion. 5 albumin. The so-prepared solution was then poured, with constant stirring, into a comparatively large volume of boiling water acidi- fied with acetic acid. By this treatment the albumin was coagulated in fairly fine flocks, after which it wras collected on a cloth filter, washed thoroughly with boiling water and pressed as dry as pos- sible. The digestions were carried out as follows: each digestive mix- ture contained 0-5 gram papoid, 10 grams of the moist coagulated egg-albumin, and 25 c. c. of water in which were dissolved the neces- sary amounts of alkali or acid to give the indicated percentages. All the mixtures of the series were placed in a water-bath kept at a temperature of 40-45° C., where they were allowed to stand for 12 hours with frequent stirring. At the end of this time, the undi- gested residues were filtered off on weighed filters of pure washed Swedish filter paper, which had been previously dried at 110° C. in suitable weighing bottles. The undigested residues were then washed with hot water until all soluble products were removed, after which they were dried at 110° C. until of constant weight. To each digestive mixture was added, likewise, eight drops of an alcoholic solution of thymol, in order to prevent possible putrefac- tion. The 10 grams of moist coagulated egg-albumin used in each digestion contained 1 *5143 grams of dry proteid, as was ascertained by drying a portion at 110° C. until of constant weight. The following figures show the results obtained: Reaction. Weight of undigested residue. Coagulated albumin digested. Neutral 0-6503 gram. 57-0 per cent.* 005 per cent. Hydrochloric acid 0-6297 “ 58-4 “ o-io 0-9060 “ 40-1 “ 2*00 “ Bicarb, soda 0-4144 “ 72-6 “ 4-00 “ “ “ 0-3896 “ 74-2 “ Without papoid, the above percentages of acid and alkali have little action on coagulated egg-albumin, as is seen from the two following experiments which were carried out exactly like the fore- going, omitting only the papoid. Reaction. Weight of undiseolved residue. Coagulated albumiu dissolved. 0-1 per cent. Hydrochloric acid 1-4990 grams. 1-0 per cent. 4 0 “ Bicarb, soda 1-4684 “ 8-0 “ * Calculated on the amount of dry albumin contained in the 10 grams of moist coagulum. Trans. Conn. Acad., Vol. IX. December, 1892. 6 JR. H. Chittenden—Papoid-digestion. From these results, it is plain that papoid will digest coagulated egg-albumin in neutral, acid and alkaline solutions ; its solvent power being most marked, in this case, in the presence of 2 to 4 per cent, sodium bicarbonate. In considering these quantitative results, and those which follow, it must be noted that the figures given cannot be taken as an exact measure of the extent of digestion, but simply as a measure of the conversion of the insoluble proteid into soluble products. Thus, it may be that the so-called undigested residue consists in some cases, wholly or in part, of alteration products, which though insoluble are still products of the activity of the ferment, while in other cases the residue may consist simply of the unaltered proteid. Dis- cussion of this point, which has a bearing on the completeness of papoid digestion, must be reserved until we come to consider the products of digestion. b. Action on Cooked Beef Proteids. The beef proteids used in this series of experiments were prepared by taking lean, round steak, passing it through a hashing machine, then washing it repeatedly with water until all blood was removed and it had become nearly white in color. It was then placed in fresh water and heated until the water boiled, after which it was strained off through a cloth filter and pressed as dry as possible. ■ Each digestive mixture contained 0-5 gram papoid, 10 grams of the cooked beef, and 25 c. c. of water in which were dissolved the indicated percentages of acid and alkali. The 10 grams of cooked beef contained 3-7438 grams of dry pro- teid (dried at 110° C.) The digestions were all warmed at 45° C. for 5£ hours, with fre- quent stirring, after which the undigested residues were filtered off, washed, dried and weighed as described in the preceding experi- ment. Reaction. Neutral 0-05 per cent. Hydrochloric acid Weight of undigested residue. 1-7782 grams. 1-7221 Cooked beef proteids digested. 52 5 per cent. 540 o-i a << 1-9179 48-7 a 0-2 a a 3 0679 18-0 < < 2-0 Bicarb, soda 1-4338 61-7 i 6 4-0 << ii 1-2651 66-2 < 6 0-2 Without papoid. Hydrochloric acid 3-5738 4-5 i 6 4-0 Bicarb, soda 3-5205 5-9 a R. H. Chittenden—Papoid-digestion. 7 From these results, it is manifest that papoid will digest and dissolve cooked beef proteids more readily even than it dissolves coagulated egg-albumin, since the above results were obtained in hours digestion at 45° C., while the slightly higher results given for the coagulated albumin were obtained after 12 hours digestion at the same temperature. It is further noticeable that the ferment acts most energetically, as in the preceding series of experiments, in the presence of 2-4 per cent, sodium bicarbonate, while a slight addition of acid increases the solvent action a trifle over that of the neutral solution. c. Action on Raw Beef Proteids. The beef used in this series of experiments was simply hashed, lean beef, washed with water until it was completely free from all soluble matters and nearly or quite white in color. Each digestive mixture contained 0*5 gram papoid, 10 grams of the prepared beef, and 25 c. c. of water containing the percentages of acid and alkali indicated. The 10 grams of beef contained 2*8508 grams of dry proteid (dried at 110° C.) The digestions were kept at 45° C. for 1 hours. Reaction. Weight of undigested residue. Raw beef proteids digested. Neutral 0-8988 gram. 68-4 per cent. 0-05 per cent. Hydrochloric acid 0-8910 “ 68-7 0-10 • kept at 45° C. ) 3-2286 “ 9-1 Neutral Solution. Alkaline Solution. Temperature. Weight of undigested residue. Proteid digested. 20° C. 1-4776 grams 58-4 per cent. 40 1-2796 68-9 “ 45 1-R054 “ 63-2 “ 60 1-0080 “ 71-6 70 Solution first ) 1-1081 “ 68-8 boiled and then - kept at 45° C. ) 2-4708 “ 30-4 Acid Solution. Temperature. Weight of undigested residue. Proteid digested. 20° C. 2-2153 grams 37 6 per cent. 40 1-4695 “ 58-6 45 1-5240 “ 57-1 60 1-2184 “ 65-7 70 Solution first 1 1-0804 “ 69-5 boiled and then V kept at 45° C. ) 3-1560 “ 11-1 The results brought out by these three series of experiments can be more readily seen by a direct comparison of the percentages digested under the different conditions, as shown in the following table : R. IF. Chittenden—Rapoid-dif/estion. 17 Proleld dixested. Temperature. Neutral sol. Acid sol. Alkaline sol. 20° C. 86 8 per cent. 376 per cent. 58-4 per cent. 40 58-5 58-6 63-9 45 55-9 57-1 63-2 60 66-8 65-7 71-6 70 Solution first t 66-3 69-5 68-8 boiled and then v kept at 45° C. ) 9-1 111 30-4 We notice first in these results a repetition of what has been already demonstrated, viz : that in an acid solution (boracic acid), papoid is slightly more active than in a neutral fluid, while in an alkaline solution (sodium bicarbonate) the activity of the ferment is increased very greatly. We now see that this statement holds good practically for all temperatures. The most striking facts, however, brought out by these experiments are : first, the marked activity of the ferment at the comparatively low temperature of 20° C. (the temperature of the room at the time the experiment was tried), especially in an alkaline fluid ; and secondly, the retention of proteolytic power after the solution of the ferment has been actually boiled. Here, too, the alkaline solution appears to exert a certain protective influence upon the ferment, which is difficult to explain. Certainly, sodium bicarbonate alone will not dissolve a coagulated proteid to any great extent, as has been already demonstrated in connection with other experiments. Hence, we are forced to the conclusion that in an alkaline fluid especially, papoid is extremely resistant to the inhibitory effects of low and high temperatures, so characteristic of most known ferments. This being true, it is ob- vious that papoid in the presence of sodium bicarbonate possesses special advantages in cases where it is desired to soften or digest tissue or other proteid matters, at comparatively low temperatures. In view of the importance of this fact, a duplicate experiment was tried, in which the digestive action of papoid was again tested at the room temperature C.) on cooked beef proteids, in a neutral solution, and in the presence of boracic acid and sodium bicarbonate. The 10 grams of prepared beef contained 3'7550 grams of dry proteid (dried at 110° C.). The digestions were con- tinued for 6 hours, at the given temperature. Reaction, Neutral Weight of undigested residue. 2•4053 grams Proteid digested. 35-9 per cent. 2-0 per cent. Boracic acid 2-2394 “ 40-3 “ 2-0 ‘ ‘ Bicarb, soda 1-9311 “ 48-5 “ 2-0 Without papoid. per cent. Bicarb, soda 3-6161 “ 3-6 “ 18 R. II. Chittenden—Papoid-digestion. Here, the same results are to be seen as in the preceding experi- ment, although the difference between the acid and the alkaline digestion is not quite as pronounced. Still, the results certainly warrant the conclusion already advanced, that in papoid we have a proteolytic agent especially adapted for the digestion of proteid matter at comparatively low temperatures. At the same time, it is a ferment very resistant to the ordinary destructive effects of high temperatures, and is especially characterized by exhibiting its maxi- mum digestive power at about 70° C. c. Influence of various Therapeutic Agents and other Substances on the Proteolytic Action of Papoid. In this connection, those substances have been chosen which might naturally be combined with papoid in its application as a therapeutic agent, either internally or externally, or which might perchance exert some modifying influence upon the action of the ferment as a general proteolytic agent. The experiments have been conducted in the same manner as those already described ; each digestive mix- ture containing 0-5 gram papoid, 10 grams of prepared beef pro- teids, raw or cooked, and 25 c. c. of water, together with the speci- fied percentage of the substance to be tested ; the digestive action in each case being compared with that of a control digestion com- posed of papoid, proteid, and water alone. Salicylic Acid. The mixtures were warmed at 45° C. for 8 hours. The 10 grams of raw beef proteids contained 2-743 grams of dry proteid (110° C.). Salicylic acid. Weight of undigested residue. Proteid digested. 0 (neutral) 1-1070 grams 59-6 per cent. 0-1 per cent. 0-9536 65-2 4 4 0-2 0-9283 “ 66-3 4 4 From these results, it is evident that salicylic acid in small quanti- ties tends to increase the proteolytic action of papoid over that of a neutral solution. As is well known, a 0’2 per cent, solution of sali- cylic acid is amply strong to act as an efficient antiseptic, prevent- ing the appearance of putrefaction in an organic fluid, even under the most favorable circumstances for its development. Consequently, salicylic acid and papoid might well be combined where application of the ferment to morbid or suppurating growths is desired. H. II. Chittenden—Papoid-digestion. 19 Carbolic Acid. The mixtures were warmed at 45° C. for hours. The 10 grams of raw beef contained 2'8508 grams of dry proteid (110° C.). Carbolic acid. Weight of undigested residue. Proteid digested. 0 (neutral) 0-9538 grant 66-5 per cent. 0-5 per cent. M441 “ 59-8 6 6 1-0 1-2270 “ 56-9 < i Hence, carbolic acid inhibits slightly the proteolytic action of papoid, but not to any great extent ; the ferment will still act vigor- ously, even in the presence of TO per cent, of the acid. Mercuric Chloride. The 10 grams of prepared raw beef contained 2’6996 grams of dry proteid (dried at 110° C.). The mixtures were warmed at 45° C. for hours. Mercuric Chloride. Weight of undigested residue. Proteid digested. 0 0-9758 gram 63-8 per cent. 0-05 per cent. 1-0660 “ 60-5 In another similar experiment, but with 0*1 per cent, mercuric chloride, 57-3 per cent, of the proteid matter Avas dissolved, while in the control digestion 59-6 per cent. Avas converted into soluble pro- ducts. Hence, mercuric chloride or corrosive sublimate, Avhen present in a neutral solution of papoid to the extent of 0-1 per cent., does not materially interfere with the proteolytic action of the fer- ment. This seems somewhat remarkable, and in conjunction with the tAVO preceding experiments makes clear tlfat papoid, as a proteo- lytic agent, is not checked to any extent in its digestive action by' three of the best known antiseptics. Chloroform. The 10 grams of prepared raw beef contained 2'743 grams of dry proteid (110° C.). The mixtures were warmed at 45° C. for 8 hours. Chloroform. Weight of undigested residue. Proteid digested. 0 1-1070 grams 59-6 per cent. 4*0 per cent. 1-2013 “ 56-2 0 0 9758 gram 63-8 8 0 per cent. 1-3423 “ 50-2 In the second experiment, with 8 per cent, of chloroform, the two mixtures were warmed at 45° C. for 7 hours, and the 10 grams of 20 H. H. Chittenden—Pcipoid-digestion. raw beef proteids contained 2-6996 grams of dry proteid. Obvi- ously, in these experiments a portion of the chloroform evaporates during the seven or eight hours, but the greater portion remains to exert such influence as it possesses. The results show some inhibi- tion of ferment action, but it is not very pronounced unless the amount of chloroform is raised to more than 4 per cent. Thymol. The 10 grams of prepared raw beef proteids contained 2’8508 grams of dry proteid (110° C.). The two mixtures were warmed at 45° C. for 7 hours. Medium. Weight of undigested residue. Proteid digested. Water 0-8988 gram 68-4 per cent. Water and thymol 1-0250 “ 64-0 A few drops of thymol solution (20 per cent, thymol in alcohol) were added to the one digestive mixture from time to time, in such quantity that there was always a strong odor of thymol, and a thin film of the substance floating on the top of the fluid. As is well known, thymol is widely used in artificial digestion experiments, to prevent possible putrefaction, as it, like chloroform, ordinarily exerts only a minimal interference with the action of the unorganized fer- ments or enzymes. The above result shows that thymol has only the same slight inhibitory effect on papoid digestion. Hydrogen Peroxide. There is no very satisfactory way of measuring the exact influence of hydrogen peroxide on the digestive action of papoid, or any other like ferment. The only way available was to dissolve the 0*5 gram of papoid in 25 c. c. of the peroxide solution (Marcliarid’s), and then to add the 10 grams of prepared raw beef. Naturally, the peroxide solution produced an immediate and pronounced change in the character of the proteid, and doubtless such diminution of diges- tive action as is apparent from the result is attributable to an altera- tion in the proteid, rather than to any change in the ferment itself. The experiment may therefore well be taken as illustrative of the extent to which a comparatively large amount of hydrogen peroxide will convert the easily digestible proteids of raw tissue into more difticultly digestible products. A control experiment, with 25 c. c. of the peroxide solution alone, shows that a small amount of the raw proteid is dissolved by this agent itself. 7t. II. Chittenden—Papoid-digestion. 21 The 10 grams of prepared raw beef contained 2'7071 grams of dry proteid (dried at 110° C.). The mixtures were kept at 40-45° C. for 7 hours. Medium. Weight of undissolved residue. Proteld dissolved. Papoid & water 0-9828 gram 63-6 per cent. Papoid & hydro- ) gen peroxide. ( 1-3812 “ 48-9 Hydrogen per- ) oxide alone. f 2-5051 “ 7-4 With cooked beef proteids, hydrogen peroxide so alters the char- acter of the material, that papoid cannot exert any solvent action whatever upon it, and the peroxide solution alone is not able to dis- solve any of the altered proteid. The 10 grams of raw beef proteids contained 2‘707 grams of dry proteid (110° C.). The mixtures were warmed at 45° C. for hours. Potassium Chlorate. Potass. Chlorate. Weight of undigested residue. Proteld digested. 0 0-7175 gram 73 4 per cent. I/O per cent. 0-8245 “ 69-5 “ 2-0 0-7948 “ 70-6 Here, we see a very slight inhibitory action, so slight as to have little significance. Practically, potassium chlorate is without any marked hindering action on papoid digestion. Sodium Chloride. With this salt, three distinct series of experiments were tried, in order to ascertain its influence on the activity of the ferment in neutral, acid and alkaline fluids. The proteid material employed was prepared cooked beef, 10 grams of which contained 3'7815 grams of dry proteid (dried at 110° C.). All of the mixtures were warmed at 45° C. for hours. Sodium chloride. Weight of undigested residue. Proteid digested. 0 1 *9017 grams 49-7 per cent. I/O per cent. 1-8091 52-1 2-0 1-7596 “ 53-4 4-0 1-7853 “ 52*7 a. In neutral solution. 22 R. II. Chittenden—Papoid-digestion. b. In alkaline solution, 2'0 per cent, sodium, bicarbonate. Sodium chloride. Weight of undigested residue. Proteid digested. 0 16233 grams 57-7 per cent. 1-0 per cent. 1-6379 “ 56-6 2-0 1-6779 55-6 4-0 1-7450 53-8 c. In acid solution, 0'1 per cent, hydrochloric acid. Sodium chloride. Weight of undigested residue. Proteid digested. 0 2-7567 grams 27‘1 per cent. 1-0 per cent. 2-2862 “ 39-5 2-0 2-2780 “ 39-7 4-0 “ 2-3844 “ 38-2 The 10 grams cooked beef proteids contained 3*7767 grams of dry proteid (110° C.). d. In acid solution, O'2 per cent, hydrochloric acid. Sodium chloride. Weight of undigested residue. Proteid digested. 0 3‘2872 grams 12-9 per cent. 1-0 per cent. 31226 17-3 « i 2-0 “ 3-1138 “ 17-5 i i 4-0 “ 3-1023 17-9 i i Sodium chloride. Neutral solution. 2 0 p. c. Bicarb, soda. 0-1 per cent. HC1. 0‘2 per cent. HC1. 0 49'7 per cent. 57-7 per cent. 27'1 per cent. 12 9 per cent. l'O per cent. 521 56-6 39 5 17-3 2-0 53-4 55-6 39-7 17-5 4-0 52-7 53-8 38-2 17-9 Percentages of Proteid Digested. From these results, it is plain that sodium chloride or common salt increases slightly the solvent action of papoid on coagulated pro- teids in neutral solutions ; while in an acid solution, 0*1 per cent, and 0*2 per cent, hydrochloric acid, it increases very greatly the solvent power of the ferment. This action of the salt we shall see later is connected with a certain solvent power on one or more of the pro- ducts of digestion, especially formed in acid solutions of papoid. Salt by itself, or in connection with dilute acid, has practically no power of dissolving the proteids of coagulated beef. In fact, the presence of salt diminishes decidedly the ordinary solvent action exerted by dilute acid alone. Thus, 0-2 per cent, hydrochloric acid by itself, at 45° C., will dissolve about 4-6 per cent, of proteid mat- ter from the 10 grams of coagulated beef in hours ; while in the presence of 2‘0 per cent, of sodium chloride, acid of the same strength, under like conditions, will dissolve only 2*6 per cent, of the proteid. The salt counteracts the swelling action of the dilute acid and thus diminishes its direct solvent power. R. H. Chittenden—Papoul-digestion. 23 In an alkaline solution of papoid, salt appears to inhibit very slightly the proteolytic action of the ferment. Bismuth Sub-nitrate. The 10 grams of prepared raw beef contained 2-707 grams of dry proteid (110° C.). The mixtures were warmed at 45° C. for 7 hours. BUmutli sub-nitratp. Weight of undigested residue. Proteid digested. 0 (Neutral) 0-9043 gram 66-5 per cent. 10 per cent. 1-4816 “ 45-2 2-0 1-6324 “ 39-6 This salt, as is evident from the results, diminishes quite decidedly the proteolytic action of the ferment, although digestion will still go on even when the salt is present in large excess. Bismuth Ammonium Citrate (soluble). The 10 grams of raw beef contained 2'2628 grams of dry pro- teid (110° C.). The mixtures were warmed at 45° C. for 8 hours. Bismuth salt. Weight of undigested residue. Proteid digested. 0 (neutral). 0 5468 grain 75 8 per cent. l’O per cent. 1-2583 “ 44-3 “ 2-0 1-2164 “ 46-2 4-0 “ 1-0027 “ 55-6 “ Here, as with bismuth sub-nitrate, we see a decided falling off in digestive power in the presence of the bismuth salt. The apparent slight increase in digestive power coincident with the increase in the percentage of the salt is probably due to the somewhat greater alkalinity, which presumably counteracts, in part, the inhibitory action of the bismuth portion of the salt. The 10 grams of raw beef proteids contained 2-6996 grams of dry proteid (110° C.). The mixtures were warmed at 45° C. for hours. Peppermint Oil. Peppermint oil. Weight of undigested residue. Proteid digested. 0 (Neutral) 0-9758 gram 63-8 per cent. 4-0 per cent. 1-0433 “ 61-3 “ Obviously, the above percentage of oil was not wholly dissolved, but it insured a fairly large excess present throughout the experi- 24 11. 11. Chittenden—Papoid-digestion. ment. This, as seen from the result, did not materially interfere with the digestive action of the ferment. Strychnin and Brucin Sulphates. The 10 grams of prepared raw beef contained 2,V0V grams of dry proteid (dried at 110° C.). The mixtures were warmed at 45° C. for hours. Alkaloid salt. Weight of undigested residue. Proteid digested. 0 0-7175 gram 73-4 per cent. 0-25 per cent. Strych. sulph. 1-2006 “ 55-6 0-50 < < 0-9089 “ 66-4 0-25 “ Brucin sulph. 0 8461 “ 68-7 “ 050 < i a 0-8068 “ 70-1 These results show that the two alkaloidal salts have a tendency to diminish the digestive power of papoid; a tendency which is more pronounced with strychnin than with brucin, the inhibitory action in the latter case being comparatively slight. Doubtless, the in- crease in digestive action coincident with the increase in the per- centage of the alkaloids is due to the slight acidity of the salts, which presumably overcomes in part the retarding effect of the alkaloidal base. Antipyrin and Acetanilid. The 10 grams of prepared raw beef contained 2’6996 grams of dry proteid (110° C.). The mixtures were warmed at 45° C. for 6£ hours. Medium. Weight of undigested residue. Proteid digested. Control 0-9758 gram 63 8 per cent. 1 '0 per cent. Antipyrin 0-9500 “ 64-8 “ 2-0 1-0803 “ 59-9 4-0 1-2992 “ 51-8 “ l'O “ Acetanilid 0-9102 “ 66-2 “ 2-0 1-0497 “ 61-1 These two drugs agree in producing a very slight increase in proteolytic action when present in the digestive mixture in moder- ate amounts, followed in the case of antipyrin by a decreased diges- tive action when the amount present reaches TO per cent. Quinine Sulphate. With this salt, there is, as the results show, a slight inhibitory action on the digestive power of the ferment, but not sufficient to materially interfere with its proteolytic action. 7t. H. Chittenden—Papoid-digestion. 25 The 10 grams of raw beef used in this experiment contained 2*707 grams of dry proteid (110° C.). The mixtures were warmed at 45° C. for 7 hours. Quinine sulphate. Weight of undigested residue. Proteid digested. 0 0-9043 gram 66-5 per cent. 1-0 per cent. M832 “ 56-2 2-0 1-0026 “ 62-9 In view of the results obtained in the majority of the preceding experiments, it would seem that papoid is characterized by a fair degree of resistance towards the usual inhibitory action of many common therapeutic agents. Certainly, the foregoing results show that papoid is able to exert its ordinary proteid-digesting power under many diverse conditions, a fact which gives it added value as a therapeutic agent. 4.— The products of papoid digestion. The foregoing experiments, taken collectively, testify to the pro- teid-dissolving power of papoid under a great diversity of condi- tions. They do not, however, show that this solvent power is neces- sarily akin to, or identical with, that of the ordinary digestive ferments. As is well known, the latter agents exert their solvent action by virtue of certain chemical changes they induce, as a result of which new and for the most part soluble products result, of which the proteoses, or albumoses, and peptones are the principal representatives. It is to be presumed, however, that papoid acts in a similar manner. Indeed, Martin* long ago pointed out that the proteolytic ferment of papaw juice, acting on blood-fibrin, formed large quantities of peptone, together with leucin and tyrosin, as products of its digestive action. Such experiments as I have tried bearing on this point clearly show that the proteid-dissolving power of papoid is due to a genuine ferment action, whereby soluble prod- ucts are formed which, so far as ordinary chemical reactions will show, are closely akin to, or identical with, those formed in gastric and pancreatic digestion. Leucin and tyrosin are likewise formed, thus showing in another way the resemblance of this ferment to the trypsin of the pancreatic juice. While, in a general way, the final products of papoid digestion are essentially the same under all ordinary circumstances, certain minor differences appear in the primary or side-products, coincident with * Journal of Physiology, vol. v, p. 225. 26 It. H. Chittenden—Papoid-digestion. changes in the reaction of the digestive fluid, and in the nature of the proteid undergoing digestion. Some of these points may be briefly summarized. In the digestion of coagulated egg-albumin with an alkaline (2'0 per cent, sodium bicarbonate) solution of papoid, even when the digestion has been long continued (say 18 hours) at a favorable tem- perature, and the ferment solution strong, there invariably remains a fairly large undissolved residue. At first glance, this would naturally appear to be simply a residue of unaltered, coagulated albumin. On being tested, however, it is found soluble, at least in great part, in w'arm (P2 per cent, hydrochloric acid, from which solution it is reprecipitated by addition of 0 5 per cqnt. sodium carbonate and redissolved by an excess of the alkaline fluid. This residue is, like- wise, directly soluble in warm 0'5 per cent, sodium carbonate, and reprecipitated by neutralization. These two reactions clearly indi- cate that the above residue cannot be composed of unaltered coagu- lated albumin, since this substance is wholly insoluble in dilute acid and alkali. The only plausible inference, therefore, is that the so- called undigested residue in this case is composed of an albumose-like body insoluble in 2 0 per cent, sodium bicarbonate, a possible primary or side-product of the papoid digestion of coagulated egg-albumin. It is evidently a somewhat unique body, differing from hetero- albumose, and from ordinary globulin, by being insoluble in salt solu- tion. Aside from this peculiar insoluble body, the other products of digestion isolated in the single experiment tried were a deutero- albumose, a fairly large amount of peptone and some leucin and tyrosin. Only a trace of protoalbumose was found, and no hetero. Neutralization of the clear, alkaline digestive fluid failed to give any precipitate, as did also boiling the neutralized solution. In fact, all of the ordinary primary products of digestion seemed in this case to be replaced by the above described insoluble albumose, compos- ing the so-called undigested residue. Of the soluble products, deuteroalbumose and peptone predominated. In the digestion of raw blood-fibrin with a neutral solution of papoid, a somewhat different condition of things was observed. The undissolved residue contained, perhaps, a small amount of the body so characteristic of the digestion of coagulated egg-albumin, but certainly not a large amount. The clear, filtered digestive fluid, however, gave evidence of the presence of a peculiar body which was wholly wanting in the digestion of the coagulated albumin. Thus, the addition of water to the clear neutral fluid gave P. II. Chittenden—Papoid-digestion. 27 a heavy white precipitate, of what was later proved to he an albu- mose-like body, readily soluble in a little 10 per cent, salt solution. Addition of 0*2 per cent, hydrochloric acid, likewise, produced a heavy precipitate of the same body, easily soluble in a slight excess of the acid. Boiling the neutral solution also gave rise to a heavy precipitate or coagulum, apparently the same body as that precipitated by water and by dilute acid, slowly but com- pletely soluble in warm 0'2 per cent, hydrochloric acid, and in warm 0'5 per cent, sodium carbonate-solution. Hence, this body is a solu- ble albumose and not a coagulable globulin. It is completely pre- cipitable from a neutral solution by heat, and partakes of the general character of heteroalbumose, being insoluble in water but com- pletely soluble in salt solutions, as well as in dilute acid and alkali. In addition to this peculiar primary product of digestion, there was also found a large amount of more soluble primary and secondary albumoses, together with true peptone, leucin and tyrosin. Raw blood-fibrin, digested with a weak hydrochloric acid (0‘05 per cent.) solution of papoid, yields the same products as those just described; the peculiar primary albumose making its appearance here in fully as large quantity as in the neutral digestion and apparently taking the place of acid-albumin, which in the single experiment tried appeared to be entirely wanting. In the digestion of cooked beef proteids with a neutral solution of papoid, as likewise with an alkaline solution of the ferment, the peculiar heteroalbumose-like body above described was wholly absent ; only the ordinary primary and secondary albumoses were observed, together with a large amount of peptone and some leucin and tyrosin. The above results, therefore, plainly warrant the statement that the power possessed by papoid of dissolving various forms of pro- teid matter is dependent upon an ordinary digestive action akin to, or identical with, that of digestive ferments in general, whether animal or vegetable. 5.—Action of Papoid on Milk. The action of papoid on milk is twofold. First, under suitable conditions, it brings about a curdling of the milk or separation of the casein, more or less complete according to the circumstances. This is followed by the ordinary digestive action of the proteolytic ferment, in which the precipitated casein is gradually converted, wholly or in part, into soluble products. 28 R. IT. Chittenden—Papoid-digestion. The act of curdling, like the process of digestion, is modified more or less by the conditions under which the experiment is tried. Thus, under some circumstances the curdling takes place quickly and the separation of the casein is quite complete. Under others, the curdling takes place slowly and is very incomplete. These points are well illustrated by the following experiments : Each mixture had a total volume of 100 c. c., composed of 25 c. c. of milk, either fresh or boiled as indicated, 25 c. c. of an aqueous solution of papoid (0'5 gram papoid), and 50 c. c. of water containing sodium bicar- bonate as indicated, or else an equal volume of lime water and water as specified. Some of the mixtures were kept at a tempera- ture of 40-45° C., while others were allowed to stand at the room temperature, viz : 22-24° C. Following, are the results obtained under the different conditions : At 40-45° C. Character ot the milk. Reaction of the mixture. Time of curdling. Boiled Neutral 3 minutes Fresh ££ 10 £ £ 10 per cent, lime water 11 ££ 20 “ “ 12 £ £ 2’0 “ Bicarb, soda* 19 1-0 “ 35 0-5 “ 55 “ Boiled 2-0 “ 240 Iu the presence of 2'0 per cent, sodium bicarbonate, the boiled milk was very incompletely curdled ; apparently, the digestion of the casein was quite advanced before any sign of separation could be observed. In the neutral solution, on the other hand, the curd- ling of the boiled milk took place almost immediately, as noted, and was at the same time very complete, the casein separating as a fine flocky precipitate, leaving an almost clear fluid. On longer stand- ing at 40° C., the separated casein was, however, • gradually dis- solved. In the presence of 0‘5 and TO per cent, sodium bicarbonate, the curdling of the fresh milk was not as complete as when 2*0 per cent, of the bicarbonate was present. This would naturally be ex- pected, since the longer the curdling is delayed the less unaltered casein will there be to separate. In all of the above cases where the curdling took place inside of 20 minutes the separation of the casein was fairly complete. * The percentages refer to the total amount of bicarbonate, or other substance, con- tained in the 100 c. c. of fluid. R. 11. Chittenden—Papoid-digestion. 29 Character of the milk. Reaction of the mixture. Time of curdling. Boiled Neutral 12 minutes. Fresh (( 145 “ < < Boiled Fresh 20 per cent, lime water j << it 2‘0 “ Bicarb, soda j J Not curdled at the end of 4 hours. At 22-24° C. It is thus evident, from the above experiments, that the curdling of milk by papoid is greatly modified by the temperature of the fluid. It is not to be assumed, however, that while precipitation of the casein is delayed by a low temperature, digestion is equally retarded. Such is certainly not the case, for digestion of the casein, whether still in solution or precipitated by the curdling process, unquestionably goes on, although naturally at a slower rate than at a higher temperature. In other words, the rate at which curdling is produced is not necessarily to be taken as a measure of the prob- able rate of proteolytic action on the proteids of the milk. The two processes are, without doubt, wholly independent, and in the case of predigesting milk, where naturally a smaller proportion of papoid is used than in the above experiments, the necessary diges- tion is accomplished without any accompanying separation of the casein; whether it be carried on at a low temperature, or at a tem- perature of 45° C.; and in the presence of water alone, or in the pr esence of sodium bicarbonate, or lime water. Hence, in order to curdle milk, fresh or boiled, a fairly large pro- portion of papoid must be employed, while for the partial digestion of milk a far smaller proportion will accomplish the desired result, and that without necessarily causing any preliminary separation of the casein. From this we may infer either that the proteolytic fer- ment is present in larger quantity in papoid than the rennet-like ferment, or else that it is far more active than its neighbor, the milk-curdling ferment. In predigesting milk with papoid, or any other ferment, the main object sought is the partial digestion of the casein ; this being, as is well-known, the most important proteid of milk, and at the same time the one most liable to cause trouble in the feeding of infants, and others, with weak digestion. In order to test the digestive action of papoid on this proteid, the casein was separated from fresh milk by precipitation with dilute acid, partially purified by re-solution in alkaline water, and reprecipitation with acid. It was then washed with water, and though still containing some adherent 30 R. H. Chittenden—Papoid-digestion. fat Avas reasonably pure. A series of digestions with papoid was then made in the manner already described ; i. e. each digestive mixture contained 0‘5 gram papoid, 8 grams of the moist casein, and 25 c. c. of water with the necessary amounts of sodium bicarbonate, etc., to give the indicated percentages. The mixtures were warmed at 40-45° C. for 6 hours, and when ready for filtration each was made as near neutral as possible, in order to precipitate any dissolved casein not converted into pro- ducts soluble in water. The 8 grams of moist casein contained 2’1377 grams of dry pro- teid (dried at 110° C.). Following are the results obtained : Reaction. Neutral 1-0 per cent. Bicarb, soda Weight of undigested residue. 1-5128 grams 1-2205 “ Casein digested.* 29-2 per cent. 42-9 “ 2-0 U 1-2468 “ 41-7 “ 4-0 6 6 1-8615 “ 36-3 “ 25-0 Lime water 1-2575 “ 41-1 “ 0-1 Hydrochloric acid 1-6774 “ 21-0 “ It is thus evident that papoid is able to digest precipitated casein under all the above conditions, but that, as with other proteids, digestion proceeds to the best advantage in the presence of 1-2 per cent, sodium bicarbonate. It is also to be noted that lime water constitutes a particularly good medium for the digestive action of the ferment on casein. The digestive action of papoid on milk-casein was next tested in a somewhat different manner, milk itself being used instead of the precipitated casein. In this series of experiments, each digestive mixture had a total volume of 100 c. c., composed of 25 c. c. of fresh milkf and 75 c. c. of water,\ the latter containing 0-5 gram of papoid, and in some cases the requisite amount of sodium bicarbon- ate to give the indicated percentages. The solutions were warmed at 45° C. for 6£ hours, when the undissolved casein was filtered off, washed, dried and weighed. In all of these mixtures, the papoid *The presence of adherent fat in the casein without doubt introduced some slight errors in the above results, as it was noticed when the mixtures were filtered, that some butyric acid had been developed, thus changing, for example, the reaction of the neutral solution to a distinctly acid one; but it is safe to assume that the above percentages at least approximately represent the rate of proteolytic action, under the given conditions. f In one mixture boiled milk was used, as noted. \ In one mixture water and lime water, as indicated. R. II. Chittenden—Papoid-digestion. 31 produced a separation of the casein inside of an hour, and in much the same order of time as shown in a previous experiment ; the neutral solution of boiled milk curdling within 5 minutes, while the mixture containing the smallest percentage of sodium bicarbonate curdled last, viz : in 55 minutes. In each case, the initial separation of the casein appeared quite complete, although of course some little allowance must be made for possible error in this direction. The digestive action of papoid was, however, quite apparent to the eye, the precipitated casein visibly diminishing in amount as the diges- tion proceeded. The 25 c. c. of milk yielded by precipitation with dilute acid (0'2 per cent, hydrochloric) TG143 grams of casein dried at 110° C. Following are the results obtained: Reaction. AVeight of undigested casein. Casein digested. Neutral 0-9.528 gram 42-2 per cent. “ (Boiled milk) 1-0311 “ 36-1 0-5 per cent. Bicarb, soda 0-9868 “ 38-8 “ 1-0 0-74758 “ 53-7 2-0 0-6979 “ 56-7 10 0 “ Lime water 1-2799 “ 20-7 From these results, it is to be noted that while boiled milk in' a neutral solution is more quickly curdled by papoid than fresh milk, digestion of the precipitated casein is somewhat less rapid. Further, in close agreement with the results found for precipitated casein, it is seen that digestion is most vigorous in the presence of 2-0 per cent, sodium bicarbonate. Somewhat peculiar, however, is the result obtained in the presence of lime water. Digestion in this case appears to have been remarkably slow; certainly not at all in conformity with the previous results with precipitated casein, and other proteids. It is evident, however, from all of the previous results, that papoid, especially in the presence of sodium bicarbonate, is particu- larly well adapted for predigesting milk, the casein being converted by it, as by alkaline trypsin solutions, into soluble and more or less diffusible products. 6.—Action of Papoid on Starch. In addition to the two ferments already described, viz: the pro- teolytic and rennet-like ferments, there is apparently present in papoid a third ferment ; an amylolytic one, capable of exerting some action upon boiled starch. At all events, papoid added to 32 R. II. Chittenden—Papoid-digestion. starch paste, preferably in the presence of sodium bicarbonate at 40° C., slowly converts a portion of the starch into soluble starch, and into a more soluble dextrin. This reaction, though plainly recognizable by the iodine test, is neither rapid nor very pro- nounced. It is not at all comparable in intensity to the proteolytic action, but still it does exist and implies the presence of a starch- converting ferment. The best result is obtained in the presence of 2-4 per cent, sodium bicarbonate. A neutral solution of the ferment is also active, but even 0’05 per cent, hydrochloric acid will produce marked inhibi- tion. In the latter case, however, the ferment is not destroyed, but simply checked in its action, since neutralization of the acid fluid with sodium bicarbonate (or better, making it alkaline) is fol- lowed by a renewal of the amylolytic action. Experimentally, the most satisfactory method of demonstrating the starch-converting power is to use a mixture composed of 0’5 gram papoid and 25 c. c- of a l’O per cent, starch paste, in which is dissolved 0 o gram sodium bicarbonate. So far as the writer’s experience extends, the amylolytic action is limited to the conversion of starch into soluble bodies giving little or no color with iodine, ordinary soluble starch being first formed. Little or no reducing sugar appears. V.—Probable Action of Papoid in the Body. Experiments already recorded show that papoid is active in the presence of percentages of sodium carbonate far larger than nor- mally occur in any of the secretions found in the alimentary tract. In fact, in the presence of 0-5 per cent, sodium carbonate, the reputed average strength of the pancreatic juice, the proteolytic action of papoid is slightly increased. The question naturally sug- gests itself, however, in this connection, whether the alkaline pan- creatic juice might not digest and destroy papoid, thus checking effectually the latter’s action. This important question was an- swered by trying several experiments, one of which may be profit- ably reported. An artificial pancreactic juice was prepared by warming at 40° C. 1 gram of trypsin (Fairchild’s) with 100 c. c. of a 1-0 per cent, sodium bicarbonate-solution and filtering from the undissolved residue. With this solution, two digestions were made with cooked beef proteids; one with 25 c. c. of the prepared trypsin solution alone, the other with the same quantity of trypsin solution plus 0’5 gram papoid. R. II Chittenden—Papoid-digest)on. 33 The two mixtures were warmed at 40° C. for 6 hours. The 10 gi*ams of cooked beef proteids contained 3 5707 grams of dry proteid (110° C.). Medium. Weight of undigested residue. Proteid digested. Trypsin sol. alone 1-5657 grams 56-1 per cent. “ “ with papoid 1-1041 “ 69-0 “ From these results it is evident that the two proteolytic ferments, trypsin and papoid, can work together in the same solution, the latter ferment contributing to the digestive strength of the former. At first glance, it might seem that in combining trypsin and papoid we should have, providing there is no destruction of the latter fer- ment, a proteolytic action numerically equivalent to that of the two ferments, but the concentration of the solution must be taken into account, as well as the inhibitory effects of the accumulated products of digestion, both of which cannot well help acting as a check to continued ferment action. Doubtless, with a weaker trypsin solu- tion the digestive action of papoid would be more strongly marked. Another point to be taken into account in considering the action of papoid in the intestine, is the influence of bile. Two experi- ments were therefore tried ; one with ox bile, the other with human bile obtained from a fistula. The 10 grams of raw beef proteids used in the first experiment contained 2’707 grams of dry proteid (110° C.). The mixtures were warmed at 45° C. for 7 hours. Dry ox bile. Weight of undigested residue. Proteid digested. 0 (Neutral) 0‘9043 gram 66-5 per cent. 4‘0 per cent. 1-1198 “ 58-6 Dry human bile. 0 (Neutral) 1-1070 “ 59-6 2-0 per cent. 0-9295 “ 66-1 In the latter experiment, with human bile, the mixtures were kept at 45° C. for 8 hours, and the 10 grams of prepared raw beef con- tained 2’743 grams of dry proteid (110° C.). The human bile was quite strongly alkaline, which probably accounts, in part, for the increased digestive action noticed. Both results, however, clearly show that the presence of bile offers little or no obstacle to the action of papoid in the intestinal tract. The only remaining point to be considered is the probable fate of papoid in the stomach. In this connection, it has already been demonstrated that the ferment is only slightly inhibited in its action by the presence of 0T per cent, hydrochloric acid, and that even in the presence of 0’2 per cent, hydrochloric acid it exhibits a fair 34 11. If. Chittenden—Papoid-digestion. degree of activity ; both of which results clearly favor the action of papoid in the stomach. Further, while the presence of 0T per cent, hydrochloric acid lessens somewhat the action of the ferment, the latter is not destroyed ; hence, by neutralization of the acid the inhibitory effect is overcome and the ferment springs into renewed activity when brought in contact with an alkaline medium. By long-continued warming of the bare ferment with 0-2 per cent, hydrochloric acid, there is a pronounced destructive action. This destruction, however, is accomplished by the free acid ; free, because of the lack of pro- teids present for it to combine with, a condition of things not so liable to occur in the case of a full stomach, which is naturally the time when a digestive ferment would be administered. The above statements are illustrated by the following facts : Two portions of papoid, 0m5 gram each, were warmed at 40° C. for hours with 25 c. c. of 0‘2 per cent., and 0T per cent, hydrochloric acid, respectively. The two solutions were then neutralized and eventually made slightly alkaline with sodium bicarbonate. Their digestive action was then compared with that of a like amount of fresh papoid, dissolved in the same quantity of fluid and of the same reaction. The 10 grams of cooked beef proteids contained 3-4420 grams of dry proteid (110° C.). The mixtures were warmed at 40° C. for 6 hours. Weight of Conditions. undigested residue. Proteid digested. Fresh papoid 1-7930 grams 47-8 per cent. Previously 10>1 , Hnl warmed with f u 1 per cent' 1-8425 “ 46-4 “ “ “ 0-2 2-9260 “ 14-9 “ It is thus evident that, under the above conditions, 0-l per cent, hydrochloric acid may hinder the digestive action of papoid, but will not destroy the ferment. With 0-2 per cent, hydrochloric acid, however, when the acid is free and not combined with proteid matter, there is a marked destruction of the ferment; not complete, but doubtless sufficient to interfere somewhat with its action. In the presence of an excess of proteid matter, this destructive action is not so marked. The presence of pepsin does not appear, materially, to modify the action of the dilute hydrochloric acid on papoid. I do not think that gastric juice of a given acidity has any more deleterious effect on the ferment, than acid of the same strength alone. In any event, 11. II. Chittenden—Papoid-digestion. 35 papoid will certainly exhibit marked proteolytic action in the pres- ence of OT per cent, hydrochloric acid and pepsin, although the conditions may not be favorable for the best action of pepsin. This is illustrated by the following experiments: an artificial gastric juice was prepared by dissolving some commercial pepsin (Fairchild’s) in OT per cent, hydrochloric acid, in the proportion of 0'1 gram pepsin to 25 c. c. of acid. Digestions were then made, with and without papoid, of raw and cooked beef proteids, with the following results: The 15 grams of raw beef used contained 4’0494 grams of dry proteid, while the 10 grams of cooked beef contained 3’9608 grams of dry proteid (110° C.). The digestions were kept at 40° C. for 6 hours. a. With raw beef proteids. Character of the fluid. Weight of undigested residue. Proteid digested. 25 c. c. pepsin-HCl alone 3 3626 grams 16-9 per cent. “ “ “ + ) 0-5 gram papoid f 1-4822 “ 63-3 “ b. With cooked beef proteids. Character of the fluid. Weight of undigested residue. Proteid digested. 25 c. c. pepsin-HCl alone 3-8689 grams 2-3 per cent. “ “ “ + j 0*5 gram papoid | j- 3-2124 “ 44-1 The action of papoid is prominent here simply because the condi- tions are favorable for its action, while they are not well adapted to the action of pepsin. The two points to be emphasized are, how- ever, first, that the presence of pepsin does not interfere with the action of papoid in an acid medium, where the other conditions are favorable to the latter ferment; and secondly, that any direct com- parison of the digestive action of the two ferments cannot well be made, since they act under such totally different conditions as regards dilution, reaction, etc. Further, in studying the action of acids on any ferment we have to consider not only the influence of a given percentage of acid, but must also take into account the absolute amount of acid, both in proportion to a given quantity of ferment and the proteid matter to be digested. It is hardly neces- sary to detail the several experiments that have led to this conclu- sion; one alone will suffice. The following mixtures were warmed at 40° C. for 6 hours, with 10 grams of cooked beef proteids, with results as indicated: 36 R. II. Chittenden—Papoid-digestion. Digestive mixture. Proteid digested. 100 c. c. 0'1 per cent. HC1, 0-1 gram pepsin, 0 papoid 21 4 per cent. ii ii “ “ 0'5 gram “ 21-3 “ 50 c. c. 0-1 per cent. HC1, 01 gram pepsin, 0 papoid 8’2 per cent. ii i i “ “ 0-5 gram “ 14-8 “ 25 c. c. 0-l per cent. HC1. 0'1 gram pepsin, 0 papoid 6‘8 per cent. ii ii “ 0'5 gram “ 34-7 “ It is thus seen that 0-5 gram of papoid, acting npon 10 grams of cooked beef proteids, in the presence of pepsin and 0M per cent, hydrochloric acid, will digest a reasonable amount of the proteid when the volume of the acid fluid is only 25 c. c., the pepsin action being very slight. As the volume of acid fluid is increased to 50 c. c., then the papoid action diminishes almost 50 per cent., while the pepsin action shows a slight increase. Increasing the volume of acid fluid still further, to 100 c. c., the pepsin action becomes paramount. Hence, it is evident that the rate of action of papoid in the stomach hinges mainly upon the presence or absence of an excess of free acid. With only combined hydrochloric acid present, and an excess of proteid matter and salts, a condition of things generally prevalent especially in the early stages of digestion, papoid cannot well help exerting its peculiar proteolytic power. And in this con- nection, it is to be remembered that papoid acts to the very best advantage in a concentrated fluid, in the presence of an excess of proteid matter. From the foregoing experiments, the following conclusions may be drawn : 1. That papoid is a true, soluble, digestive ferment or mixture of ferments, of vegetable origin. 2. That it has marked proteolytic action in acid, alkaline, and neutral solutions and in the presence of many chemicals, antiseptics, and therapeutic agents. 3. That it has a peculiar softening and disintegrating action on proteids, and that its general proteolytic action is that of a genuine digestive ferment, similar to the ferments of animal origin. 4. That it has a certain amount of ainylolytic, or starch-dissolving power. 5. That it has a marked rennet-like action upon milk, and a pro- nounced digestive action upon milk-casein. 6. That it exerts its peculiar digestive power at a wide range of temperatures. 7. That the ordinary conditions of health and disease in the stomach and intestine are not liable to check its action, while certain possible conditions may accelerate it.