426 HEPBURN— BIOCHEMIC.\L STUDIES 



extract from pitchers, which had received the acid treatment, had com- 

 pletely dissolved the fibrin, while the extract from pitchers not so treated 

 had but slightly attacked the fibrin. The solutions, obtained by filtra- 

 tion of the contents of each tube, always gave a positive response to the 

 biuret test. 



Vines states: — "These experiments seem to indicate that in the gland- 

 cells of the Nepenthes pitchers, . . . ., the digestive ferment exists at first 

 in combination with some other body, as zymogen — and that in plants, 

 as in animals, this zymogen can be split up by the action of dilute acid, 

 the free ferment making its appearance as a result of this decomposition. " 

 In another report on this research, Vines (25) also described tests 

 for the presence of diastase in the pitchers. The glycerol extracts of the 

 pitchers were without action on starch, therefore, did not contain dias- 

 tase. He also noted that the glycerol extract did not contain sugar. 



During a further study of the proteolytic enzyme of Nepenthes, 

 Vines (6) found that pitcher liquor plus 0.25 percent hydrochloric acid 

 completely digested fibrin in the presence of bactericides such as potas- 

 sium cyanide, chloroform, thymol. The fibrin was dissolved when mer- 

 curic chloride (approximately 0.5 percent) was used as a bactericide; 

 however, the proteolysis was somewhat retarded. Fibrin was also 

 digested by pitcher liquor to which had been added 1 drop of concen- 

 trated hydrochloric acid and sufficient hydrocyanic acid to render the 

 concentration of the latter acid one percent. The liquor plus 0.25 percent 

 hydrochloric acid partially digested coagulated egg albumen in the 

 presence of potassium cyanide or thymol, the products of proteolysis 

 being detected by the biuret test. 



The enzyme and its proteolytic power were destroyed by the action 

 of 1 percent sodium hydroxide for 1 hour, or of 5 percent sodium car- 

 bonate for three hours, at a temperature of 35 to 40° C; the solutions 

 were then neutralized, and tested for protease in the usual way, but 

 digestion of the substrate never occurred. 



The influence of the following concentrations of hydrochloric acid on 

 the proteolysis was studied:— 1%, 0.5%, 0.25%, 0.125%. The optimum 

 acidity for digestion was found to be 0.25%. 



The enzyme was isolated from 100 cc. of pitcher liquor by the follow- 

 ing procedure. An equal volume of absolute alcohol was added, then 

 phosphoric acid and lime water; the solution was neutralized with ammon- 

 ium carbonate; the precipitate was collected on a filter, and permitted 

 to drain over night. A portion of the precipitate was shaken with 10 cc. 

 of 0.25 percent hydrochloric acid, and the solution was filtered; the 



