240 



mACTIVATION OF TRYPSIN. I 



TABLE III. 

 Dialysis of Trypsin. 

 2.5 gm. of trypsin in 25 cc. water, dialyzed under pressure 18 hours, and filtered. 



pH 



Before dialysis. 

 After dialysis . . 



6.2 to 6.4 



Preparation of the Inhibiting Solution. 



Bayliss^ found that glycine and other amino-acids as well as the 

 digested protein solution inhibited the action of trypsin. Several 

 amino-acids were tried but with negative results except in concentra- 

 tion very much higher than could possibly be present from the pro- 

 tein. In such high concentration the determination becomes uncer- 

 tain, owing to the high conductivity of the solution. Bayliss' experi- 

 ments were made at a time when the determination of the hydrogen 

 ion concentration was a difficult matter and it seems possible that the 

 results he obtained were due to changes in the pH caused by the 

 addition of the amino-acids rather than to an effect on the enzyme. 

 A solution of casein or gelatin which has been hydrolyzed by trypsin 

 does show marked inhibitory effects, however. Owing to the manner 

 in which these experiments were made it was necessary to have the 

 solution nearly salt-free and in a concentrated form, so that the 

 volume change on adding it to the gelatin would be small. It is also 

 necessary to be sure that there are no products left in solution that 

 can be further acted on by trypsin. It was found that a solution 

 having strong inhibitory powers could be made from either gelatin 

 or casein by the following method. 



Preparation from Gelatin. — 3 liters of 1.5 per cent gelatin were titrated to a 

 pH of 9.0 with Ba(0H)2 and 10 cc. dialyzed trypsin and a few crystals of thymol 

 added. The solution was kept at 23° until no further increase in the formol 

 titration could be noted. The titration increases about 700 per cent. The 

 solution was then titrated to a pH of 6.3 with sulfuric acid and the barium sulfate 



