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ON THE PRESENCE OF A PROTEOLYTIC ENZYME IN THE NORMAL SERUM OF THE OX. BY S. G. HEDIN. (Department of Pathological Chemistry, Lister Institute of Preventive Medicine, Lonidon.) ALTHOUGH proteolytic enzymes on theoretical grounds have been supposed to be present in the serum, their presence was not proved by analytical means till Delezenne and Pozerski lately demonstrated that the serum by the action of chloroform at 370 can be made to act upon proteidsl. This effect they explained by supposing that the chloroform destroys antibodies, which in the normal serum prevent the enzyme from acting. Before I knew of the investigations of Delezenne and Pozerski I was able to show the presence of a proteolytic enzyme in the serum by precipitating it together with the globulin or together with nuclein substances added to the serum. The method adopted for showing proteolytic action has been to precipitate a known volume of the fluid with a known volume of tannic acid solution and estimating the nitrogen in a known volume of the filtrate2. The difference between the nitrogen obtained after and before the digestion then indicates the extent of the digestion that has taken place. In all cases in which I tested the serum by itself for proteolytic action, I obtained exactly the same amount of nitrogen before and after several days' digestion, which proves that at any rate no peptones or lower digestion products were formed. I was led to suspect that the serum might contain a proteolytic enzyme, although no action could be demonstrated directly, by the- fact that the antiproteolytic action of the serum,, as shown by myself in the case of the lieno-a-protease, is most marked in the serum albumin and less in the pseudoglobulin, whilst the euglobulin showed the least antiproteolytic action or even increased the activity of the spleen enzyme. Compt. Rend. de la Soc. biol. Vol. Lv. No. 19 (1903). 2 The tannic acid solution contained 100 grams of tannic acid, 100 grams of sodium chloride, and 60 c.c. of glacial acetic acid in one litre, 13-2

196 S. G. HEDIN. The euglobulin is the part of the serum globulins least soluble in water, and roughly it corresponds to the globuilin coming down on dialysing or on adding acetic acid to the diluted serum. I therefore have tried the digesting power of the precipitates obtained in these ways. The results are given by the following experiments Exp. 1. Globulin obtained on dialysing ox serum was dissolved in 0-25 0/0 Na2CO3 and tested as to its digesting power. The analysis was carried out by adding 10 c.c. of tannic acid solution and estimating the N in 5 c.c. of the filtrate. Before the digestion: 5 c.c. coag. serum+ 5 c.c. glob. sol. gave N corresponding to 1 0 c.c. 1n. acid. boiled serum albumin+5 c.c. glob. sol. 0 9 After 6 days' digestion: 5 c.c. coag. serum+5 c.c. glob. sol. 2-2,, boiled serum albumin+5 c.c. glob. sol. 1.9 Exp. 2. Precipitate obtained from ox serum with acetic acid was dissolved in 0-25 0/0 Na2C03 and used for digestion. Before the digestion: 5 c.c. coag. serum+5 c.c. glob. sol. 0-8 c.c. 1n. acid. After 4 days' digestion: 5 c.c. coag. serum+5 c.c. glob. sol. 1-4,,,,,,,, +0 01 gr. casein 2 6,,,, +5 c.c. glob. heated to 560+ 0 01 gr. casein 0-8,. Exp. 3. Globulin obtained from ox serum with acetic acid. The casein and the globuilin were dissolved in 0 25 0/0 Na2CO3. Before the digestion 5 c.c. coag. serum+5 c.c. glob. sol.+5 c.c. 0-25 0/0 Na2CO3 0 75 c.c. -O n. acid. casein sol. 0-8 nucleo-alb. sol. obtained from the ox spleen 1x6,, After 14 days' digestion: 5 c.c. coag. serum+5 c.c. glob. sol. +5 c.c. 0 25 0/0 NaCO3 4-6 (The coag. serum was not dissolved in 3 days.) 5 c.c. coag. serum+5 c.c. glob. sol. +5 c.c. casein 5 8,, (Serum nearly dissolved in 3 days.) 5 c.c. coag. serum+5 c.c. glob. +5c.c. spleen nucleo-alb. 10-2 (Serum dissolved in 3 days.) 5c.c.coag. serum+5c.c.glob. heated to 50 +5c.c. casein sol. 4 8,.,. Exp. 4. Globulin obtained from ox serum with acetic acid. Before the digestion: 2 grms. coag. egg-alb. + 5 c.c. glob. sol. +5 c.c. 0 25 0/0 Na2C03 1 1 c.c. 1 n. acid. spleen nucleo-alb. 19,. 5 c.c. coag. serum,,,,,, casein sol. 1-2 0-25 0/0 Na203 0 9,.,, spleen nucleo-alb. 1-8, casein sol. 10,,

ENZYME IN OX SERUM. 197 After 10 days' digestion: 2 grms. coag. egg-alb. + 5 c.c. glob. sol. + 5 c.c. 0 25 0/0 Na2CO3 1-2 c.c. fo n. acid. spleen nucleo-alb. 5-2 casein sol. 3.5 5 c.c. coag. serum,,,,,, 0-25 /0 Na2CO3 43,3. spleen nueleo-alb. 7 0 casein sol. 7-8 The enzyme had an evident effect upon gelatin. Exp. 5. Globulin obtained from ox serum with acetic acid. Before the digestion: 5 1 c.c. glob. sol. + 5 c.e. 0 25 e/0 Na2CO3 0 3 c.c. lo n. acid.,,, ),, casein sol. 0 4 to 2 grms. boiled egg-alb.,, glob. sol. +5.c. Na2CO sol. 0-35, ṫ.,,,,,,,,,, 9casein sol. 0-45 After 4 days' digestion: 5 c.c. glob. sol. + 5 c.c. 0-25 0/0 Na2CO, sol. 0 3,,,, TV casein sol 3'55 2 grms. boiled egg-alb.,, glob. sol.+ 5 c.c. casein sol. 3'25,I.. After 10 days' digestion: 5 c.c. glob. sol. +5 c.c. casein sol. 4'0,.,. 2 grms. boiled egg-alb.,, glob. sol. + 5 c.c. 0 25 e/0 Na2CO3 sol. 0 5,....,,,,,,,,, 9IIcasein sol. 4.7 Exp. 6. Globulin before digestion 0 3,...,, after 6 days' digestion 0'3,,.. On comparing the corresponding samples before and after digestion the following conclusions can be drawn: 1. The proteolytic enzyme contained in the globulin does not digest the globulin itself (Exps. 5, 6), or coagulated egg-albumin (Exps. 4, 5). 2. The enzyme acts upon casein (Exp. 5), uipon gelatin (Exp. 4), and upon coagulated ox serum (Exps. 1, 2, 3, 4). 3. The enzyme is destroyed or very nearly destroyed when its solution in 0-25 /e NasCO3 is heated to 550 for half-an-hour but not at 500 (Exps. 2, 3). Casein and spleen nucleo-albumin when added to the enzyme increase its effect, and the increase does not seem to be due only to the digestion of the substance added, since coagulated serum is more rapidly dissolved with than without the presence of a nuclein substance, as can be ascertained by inspection (Exp. 3). Whether the casein makes the enzyme act upon coagulated egg-albumin seems uncertain (Exp. 5). In Exp. 4 it was found that the globulin acted upon gelatin even without the presence of a nuclein substance.

198-1s. G. HEDIN. Delezenne and Pozerski state that serum acted upon with chloroform digests gelatin and-casein but does not digest coagulated egg-albumin, and it therefore seems very likely that their active substance is identical with the enzyme, taken down together with the globulin in my experiments. Since I bad found in my experiments with the spleen enzymes that they are readily carried down by nucleo-albumins, I thought it advisable to try to precipitate the serum enzyme together with casein. Therefore the serum was diluted with 3-4 volumes of water, and of a saturated soluition of casein in 0-25 0/0 Na2C03 (prepared at 370), 10 c.c. were added for each 100 c.c. of serum. Then the casein was precipitated by adding 0'2 0/0 of acetic acid. After filtering and washing, the precipitate was dissolved in 0-25 0/0 Na2CO. and made to digest itself. As the following experirnents will prove, the casein precipitate shows rather a strong activity in an alkaline medium. Only in one case (Exp. 1) a slight action has been observed in the presence of acetic acid. In the presence of hydrochloric acid no action has been obtained. Exp. 1. Digestion in a solution of 0:25 0/0 Na2CO.. Before the digestion the nitrogen not precipitable with tannic acid corresponded to 06 c.c. I n. acid. After 4 days' digestion 3 0,...,, 7,, 4.4,...,, 115.,, ~~~~~~~5 0,.,. Digestion with 0'2 0/0 acetic acid: After 4 days' digestion 0.8,... 7,, 1-2 IV.. 11,, 16 to Exp. 2. Before digestion 0-6 cc. 1 n. acid. After 6 days' digestion: With 0 25 0/0 Na2CO3 3-2,,..,, 0 2 /0 acetic acid 0'6,... 0-2 /0 HCl 0-6,.. After 12 days' digestion: With Na2C03 4-2,.., HCl 0.8,. Boiled sample digested 12 days with Na2CO3 0-8,I.. The experiments seem to prove that the ox serum contains one or more proteolytic enzymes, which can be precipitated together with the globulins of the serum or together with added casein. Since the serum itself shows no proteolytic action it seems probable that the enzyme in the serum is prevented from acting by the presence of antibodies. As

ENZYME IN OX SER UM. a matter of fact the casein protease digests boiled and coagulated serum far more easily than unboiled serum, as set forth by the following experiments. Exp. 1. 5 c.c. serum were digested with 5 c.c. casein protease in O`25 0/0 N;2C03 solution. After 5 days: Serum (not heated) 16e.c. j0 n. acid. Heated serum 388,,,, After 11 days: Serum (not'heated) 1-6,... Heated serum 52,... Exp. 2. Serum was digested with casein protease as in Exp. 1. Before the digestion: 1 Coagulated serum Serum (not heated) 10,... After 4 days' digestion: Serum (not heated) 1-8,... Heated serum 4,.. After 8 days: Serum (not heated) 1-8,... Heated serum 5.5 1-2 c.c. --O n. acid. 199 The result of these experiments might be explained by supposing that the checking influence of the serum upon the activity of its own enzyme disappears on heating to 1000, that is to say, the antibody is destroyed on heating. In order to make out which part of the proteids contains the antibody I tried separately the precipitate obtained on dialysing, and the euglobulin, pseudoglobulin and albumin obtained from the filtrate with Am2SO4 according to the method of Spiro'. The different fractions were dissolved in 0-25 0/0 Na2CO3, whereupon their influence upon the casein protease was tried by digesting together 5 c.c. coag. serum + 5 c.c. casein protease + 5 c.c. of the different proteid fractions. By boiling either the casein protease or the proteid fractions before digesting, the influence of the heated part could be eliminated without altering the amount of proteid present. The samples containing boiled casein protease, when compared with the samples analysed before the digestion, show whether the different proteid fractions have any 1 Zeit8chr. f. physiol. Chem. xxxi. p. 132, 1900, and xxxvi. p. 407, 1902.

200 S. G. HEDIN. proteolytic activity by themselves, and the samples with heated proteid fractions as compared with the ones containing the same proteids not heated show the influence of the different fractions upon the protease of the serum. The analysis was carried out by precipitating the 15 c.c. of fluid by adding 10 c.c. of tannic acid solution, estimating the N in 5 c.c. of the filtrate and calculating the result for the original fluid (15 c.o.). Before the digestion: 5 c.c. coag. serum + 5 c.c. protease +5 c.c. dial. pree. 0-8 c.c. 10 n. acid. 1.,,,,,, euglob. 0 7,.. I,,,,,,,, pseudoglob. 0-8,,,,,,,, albumin 0@8,... After 5 days' digestion: 5 c.c. coag. serum + 5 c.c. boiled protease+ 5 c.c. dial. prec. 37,. 2.,,,,,,,,I,,I euglob. 1-3,.. pi,,,,,,,, pseudoglob. 08,,,,,,,,,,, albumin 0'8,... 5 c.c. coag. serum+5 c.c. protease+5 c.c. boiled dial. prec. 37,,,,,,,,,,, euglob. 3*5 3 3.,,,,,,,, pseudoglob. 3.5,,,,,,,,,, albumin 3-75,,.. 5 c.c. coag. serum +5 c.c. protease + 5 c.c. dial. prec. 4-3,.,,,,,,,, euglob. 2,7 pseudoglob. 2-5,,,,,,,, albumin 1-3,... The experiments of group 2 as compared with the corresponding experiments of group 1 show that the dialysis-precipitate and to a less extent also the euglobulin in this case have a proteolytic effect of their own, which is consistent with my previous results, whereas the pseudoglobulin and the albumin show no activity at all. The different experiments of group 3, where the effect of the different proteid fractions is elimjinated, have given almost the same results, as was to be expected. When compared with the corresponding experiments of group 4 they show that the dialysis-precipitate increases the activity of the casein protease, whilst the albumin and to a less extent the pseudoglobulin and also the euglobulin check its activity. The proteid fractions had been prepared fromi the same serum portion, and the volume of the albumin was 4-5 times as large as the volumes of the other fractions. We therefore must be right in concluding that the anti-enzyme or anti-enzymes mainly are attached to the albumin of the

ENZYME IN OX SER UM. serum. In this respect the anti-enzyme of the serum protease agrees with the anti-enzyme of the a-protease of the spleen, as found by myself'. CONCLUSIONS. The results of the abovq investigations can be summed up as follows:- 1. The serum of the ox contains a weak proteolytic enzyme, which acts in an alkaline medium. The enzyme is partly brought down together with the part of the globulins precipitated on dialysing, on adding acetic acid, or on i saturation with Am2SO4. 2. The enzyme thrown down with the globulin acts upon casein, gelatin, and coagulated serum, but does not act upon the globulin itself or upon coagulated egg-albumin. It is destroyed by heating to 5a for half-an-hour. 3. The easiest way of obtaining the enzyme is to precipitate it together with a nuclein substance, e.g. casein. 4. Antibodies prevent the enzyme from acting in the serum. These antibodies are mainly contained in the albumin fraction. As to the origin of the enzyme nothing can be stated at present. Yet it should be remembered that a similar enzyme has been found in the leucocytes of the spleen, and it therefore does not seem to be impossible that the serum protease should be derived from the leucocytes in the blood or in other organs either by a destructive process, which might set the enzyme free, or by an act of secretion. 1 This Journal, xxx. p. 155. 1903. 201

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