Experimental. Schmidt, in his experiments, boiled his solutions

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1 PROTECTION OF TRYPSIN FROM DESTRUCTION BY HEAT. BY D. IL DE SOUZA. (From the Institute of Physiology, University College, London.) E. W. SCHMIDT' has recently claimed: that trypsin in the presence of peptone, gelatine, or agar, may be boiled and yet still retain its digestive power. The results as Schmidt points out have a practical application, for, if correct, they afford a means of sterilising enzytnes by heat without destroying them. At Dr Bayliss's suggestion I have made some experiments on the degree of protective action of peptone for trypsin. Methods. The trypsin used was a 50/o filtered solution of Hola(iin (Fairchild Bros. and Foster) with which Dr Plimmer kindly supplied me. It was neutral to litmuts and gave a slight precipitate on boiling. Witte's peptone was em-ployed in 5, 10 or 200/0 solution. Its reaction to litmus was slightly alkaline. In most of the experiments the digestive activity of the trypsin before and after subjection to heat was measured by estimating the amino-acid groupings present by S6rensen's2 formaldehyde method, titrating with sodium hydrate and not baryta as the alkali. Hammersten's casein (Kahlbauim), a 40/, solution in 0.40/0 sodium carbonate, was used as substrate. It gave a slightly alkaline reaction with litmus. As a rule the enzyme was added to 100 c.c. of this casein solution and a sample of 25 c.c. was withdrawn at once. The rest was put inito the incubator at 36' or 37 C. and other samples of 25 c.c. were removed after different intervals of time. The quantity of amino-acids in each of these samples was estimated. All dilutions were made with distilled water. Experimental. Schmidt, in his experiments, boiled his solutions in a test tube, heating until the whole column of liquid was in brisk ebullition, anid used fibrin as his substrate. In my earlier experiments this plan was followed. 1 Zt8ch. f. physiol. Chem. LXVII. p Biochem. Ztsch. vii. p ; xxv. p

2 PROTECTION OF TRYPSIN. 375 A series of five test tubes, A, B, C, D, E, was arranged containing A. Trypsin 1 c. c. + water 10 c.c. B. (Trypsin 1 c.c. +water 10 c.c.) boiled. C. Trypsin 1 c.c. +peptone 10 c.c. D. (Trypsin 1 c.c. +peptone 10 c.c.) boiled. E. Water 1 c.c. +peptone 10 c.c. 5 c.c. of sodium carbonate were added to each to ensure an alkaline medium for digestion, and a piece of boiled and washed fibrin was put into each tube, which was then corked and left in the incubator at 360 C. for 20 to 24 hours. The results were inconclusive as putrefaction, evidenced by an offensive odour, usually took place, so that when the odour was not present one had to be chary in deciding whether the disappearance of fibrin was due to putrefaction or to the action of trypsin. The addition of *18 c.c. of toluene to each tube before putting it into the incubator gave more constant results. After hours nearly all the fibrin in A and C was dissolved; in B anid E there was no change; in D the fibrin was broken but had not perceptibly (liminished in amount. The trypsin, then, which was active in watery or peptone solution and was destroyed by boiling in watery solution, was afforded by the presence of peptone little if any protection from this destruction. There are, however, serious objections to the method. By boiling the solutions in a test tuibe it is difficult to be sure that the boiling temperature is attained throughout, especially in the fluid clinging to the sides of the upper part of the tube. Fibrin even when boiled is too easily attacked to be a good substrate, and is useless for the accurate measurement of the rate of digestion. In all the remaining experiments small flasks were employed instead of test tubes. The solutions were introduced into and removed from them with a pipette, and care was taken to avoid contact with the neck of the flask. Heating was effected by immersing the flask in a water bath at constant temperature for a definite period. Instead of fibrin, Hammersten's casein was used as substrate. Toluene was always added as it did not apparently interfere with tryptic digestion, and its presence delayed, although it did not always prevent, putrefaction. The following series was arranged: A. Trypsin 1 c.c. +water 10 c.c. B. (Trypsin 1 c.c. + water 10 c.c.) heated at 800 C. for 5 mins. C. Trypsin 1 c.c. + peptone 10 c.c. D. (Trypsin 1 c.c. + peptone 10 c.c.) heated at 800 C. for 5 mins. E. Water 1 c.c. +peptone 10 c.c. Each mixture was added to casein solution in the ratio 1 to 10 (50 or 100 c.c. of casein being used), and toluene 1 c.c. 25 c.c. samples were taken from these at once and after

3 376 D. HI. DE SO UZA. different intervals of time, and the amino-acids estimated. The increase in amino-acids N in cc. - NaOH is shown in the following table: 10 Exp I II III IV V strength of peptone solution /, Time in hours i Increase in amino-acids in A *,,,,,, B ,,,,,, C D ,,.. E,,, The figures show that the peptone solution used (E) had no digestive activity. The normal digestive power of the trypsin in watery solution and in presence of peptone is given by A and C respectively. The watery solution heated at 800 C. for five minutes (B) has entirely lost this power, the 0-2 in the second column being withiin the limits of experimental error. On the other hand the trypsin heated in presence of peptone (D) is still able to effect a variable amount of digestion, hardly any with 50/0 peptone, but a measurable though small amount with the higher percentages. In the above table the numbers given are the results of estimations made after an interval of about 24 hours. These have been selected to render comparison easier. The experiments were usually carried on for a longer time and the results were similar, e.g. in Exp. V after 70 hours the figures were: A 20-9 D 4-6 B 0 E 0 C 24-4 but the longer the duration of the experiment the more difficult it is to exclude putrefaction as a factor from the results. The next set of experiments was performed to see whether stronger evidence of protection by peptone could be obtained at lower temperatures. The same series was arranged, but the temperature of the water bath was 65' or 70 instead of 800 C., and heating was carried on for 5, 10, or 15 mins. 20 /o peptone solution was always used, and the estimations were made after 24 hours. These results were obtained: Excp. Exp.~~ :..... VI VII VIII IX ix Temp. (C.) at which B and D were heated, Time in mins. during which B and D were heated Increase in amino-acids in A ,,,,,, B ,,,,,, C ,,,,,, D ,,,,,,

4 PROTECTION OF TRYPSIA.377 As before A and C show the digestive activity of trypsin in watery and peptone solutions respectively. A compariso'n of B with A reveals the fact that the trypsin in watery solution is not entirely destroyed at 70 C. until heated for 15 mins. In the former experiments heating at 80 C. for five mins. destroyed it, but in these later experiments there is some digestive power left after heating at 700 C. for five mins., and still more after heating at 650 C. for five mins. In all these cases the same bulk of solution was used (11 c.c.). Looking at D one finds that the trypsin in peptone solution is never entirely destroyed, and that it retains a greater digestive activity than the heated watery solution. Hence it appears that the peptone exerts a protective power as long as the heating is not sufficient to destroy completely the trypsin in watery solution, and that the greater the amount of this trypsin liable to destruction the less the protective power of the peptone. When the heating is sufficient to cause complete destruction of the trypsin in watery solution the protective power of peptone is slight. At the lower temperature 700 C. it is greater than at 800 C., but it is too small for practical application in the sterilisation of enzymes. It occurred to me, on examining the conditions of experiment, that objection might be taken to the fact that, of the solutions heated, the peptone was slightly alkaline and the watery solution neutral. Since trypsin is most easily destroyed in alkaline solution the peptone may have been acting at a disadvantage, so that its protective power may be greater than it appears to be in the above results. The point was tested by experiments in which the heated watery and peptone solutions were both alkaline or both acid, and were compared with another pair both neutral: 20 0/0 peptone was used and the solutions were heated at 80' C. for five mins. The watery trypsin was destroyed whatever the reaction. The peptone trypsin always retained a slight digestive power. No difference was observed between the acid and neutral preparations, but the activity of these was a little greater than that of the alkaline. In one case, for exatrmple, the following numbers were obtained after 24 hours digestion: Trypsin 1 c.c. +water 10 c neutral, heated 0 alkaline, heated 0 Trypsin 1 c.c. +peptone 10 c.c neutral, heated 2-5 alkaline, heated 1-7

5 378 D. H. DE SO UZA. The difference between alkaline and neutral solutions is, however, small, the numbers being of the same order of magnitude, so that even if a correction for alkalinity be made in the previous experiments the resuilts will not differ greatly from those actuially obtained-certainly not sufficiently to affect the conclusions already reached. SUMMARY. 1. At 80 C., after five mins. in the water bath, trypsin in aqueous solution is entirely destroyed. The presence of peptone in the solution protects the trypsin only slightly from this destruction, and rather more in an aci(d or neutral than in an alkaline solution. 2. At lower temperatures, e.g C., trypsin in aqueous solution is not all destroyed in five mins. Peptone protects it, but the greater the amount of trypsin in watery solution liable to destruction the less is the protective power of the peptone. 3. The piotection at the higher temperatures is too small in amount to be of any value in sterilising enzymes by heat. 4. Experiments without antiseptics are unreliable owing to the difficulty in excluding putrefaction.