444 HEPBURN— BIOCHEMICAL STUDIES 



(3) The cleavage of glycyltryptophane. 



The temperature of incubation was 37°C., unless otherwise stated. 



Formol-titration 



The following substrates were used: — ovalbumen, fibrin, edestin, 

 ovomucoid, Nahrstofif-Heyden, and Witte peptone. The fibrin was 

 prepared from ox blood; the ovomucoid was obtained by the procedure 

 of Eddy (20); the Nahrstoff-Heyden, according to Gotschlich (21), was 

 a mixture of different albumoses. 



After incubation, any insoluble protein was removed by filtration, 

 and was washed on the filter; the combined filtrate and washings were 

 made neutral to phenolphthalein. If metaprotein separated, it was 

 filtered out and washed on the filter; the filtrate and washings were 

 again made neutral to phenolphthalein — if necessary — and one-half 

 of their volume of formol (40 percent, formaldehyde), previously ren- 

 dered neutral to phenolphthalein, was added. The basic amino group 

 in the amino acid molecule was thereby converted into its methylene 

 derivative by condensation with the formaldehyde, and no longer neu- 

 traUzed the acidic carboxyl group in the same molecule. This carboxyl 

 group now functioned as an acid, giving the solution a reaction acid 

 to phenolphthalein. This acidity, due to amino acids, was immediately 

 titrated with standard fixed alkaline hydroxide, using phenolphthalein 

 as the indicator, and served as a measure of the proteolysis. Usually 0.1 iV 

 sodium hydroxide was used for the titrations; however, 0.05 N sodium 

 hydroxide was used when it was expected that the proteolysis would 

 be slight on account of the small volume of pitcher liquor used. 



The following experiments were made with the liquor from stimulated 

 pitchers. 



Ovalbumen (0.05 gram) was digested with 15 cc. of pitcher liquor 

 for 3 days. After the addition of formol, the determination proper 

 required 0.15 cc, the blank experiment 0.00 cc. 0.05 X sodium hydrox- 

 ide for the neutralization of the amino acids. 



Fibrin (0.05 gram) was digested with 15 cc. of pitcher liquor for 

 14 days; on titration after the addition of formol, the determination 

 proper required 0.45 cc, the blank 0.00 cc 0.1 N sodimn hydroxide. 



Ovomucoid (0.05 gram) was dissolved in 10 cc of water, then in- 

 cubated with 5.5 cc. of pitcher liquor for 6 days. The formol titration 

 was:- determination proper 0.10 cc, blank 0.00 cc. 0.1 N sodium 

 hydroxide. 



