448 HEPBURN— BIOCHEMIC.\L STUDIES 



In one experiment, 1 cc. of liquor, 2 cc. of the edestan solution, 

 and sufficient water to render the total volume 5 cc., were mixed; and 

 the mixture was incubated for 8 days. On neutraliztion, a voluminous 

 precipitate formed in the blank; the precipitate, which formed in the 

 determination proper, was about one -half as great as that in the blank, 

 showing that partial digestion of the edestan had occurred. 



In another experiment, a mixture of 4 cc. of pitcher liquor and 1 cc. 

 of the edestan solution was incubated for 13 days. On neutralization, 

 the blank yielded a voluminous precipitate, the determination proper, 

 a precipitate but one-tenth as great as that in the blank. Hence partial 

 digestion of the edestan had occurred. 



Protean derived from castor-bean globulin 



The castor-bean globulin, used in these experiments, was presented 

 by Dr. Isaac F. Harris, to whom I am also indebted for the outline of 

 its preparation. Ground castor-beans were extracted with gasoline 

 to remove the oil, then were extracted with a 10 percent, sodium chloride 

 solution. This solution was filtered, and the clear filtrate was dialyzed. 

 The globulin, which deposited, was dissolved in a 10 percent, solution 

 of sodium chloride; the solution was filtered and dialyzed. The globuUn, 

 which separated, was washed with water, alcohol, and ether, and was 

 desiccated. 



A 2 percent, solution of this globuhn in a 5 percent, solution of sodium 

 chloride was used as a reagent for proteolytic enzymes; the solution 

 was filtered, if necessary. When the clear solution of the globulin was 

 mixed with the pitcher hquor and 0.5 cc. of 0.1 N hydrochloric acid was 

 added, a cloudy precipitate of the protean derived from the globulin 

 formed. On incubation, if a proteolytic enzyme, active in the presence 

 of hydrochloric acid, was present, the insoluble protean was digested 

 and converted into less complex, soluble compounds, which dissolved; 

 and the cloud gradually became less dense, and finally disappeared. 



The following experiments were made on liquor from stimulated 

 pitchers. 



The pitcher liquor (2.5 cc.) was incubated with 2 cc. of the globulin 

 solution and 0.5 cc. 0.1 N hydrochloric acid. Proteolysis was marked 

 on the third day, advanced on the seventh day, and ahnost complete 

 on the twelfth day. 



The experiment was repeated using 0.5 cc. of pitcher liquor, 4 cc. of 

 the globulin solution, 1 cc. 0.1 .Y hydrochloric acid, and 4.5 cc. of water 

 (to secure the same concentration of substrate and of acid as in the pre- 



