JOHN H. NORTHROP 59 



hydrolyzed. It is, therefore, not possible to conclude that the enz>'me 

 ceases to act, when a certain stage of the hydrolysis has been reached, 

 because all the linkages which it is capable of hydrolyzing have already 

 been split. The molecule may have been modified in some other 

 way and so become resistant to the action of the enzyme although 

 still containing a linkage which under certain conditions may be 

 attacked. The same influence is undoubtedly also shown on the rate 

 of hydrolysis by acids or alkali. There does not seem to be any evi- 

 dence to distinguish qualitatively between the specificity of an enzyme 

 and of hydrogen ions. 



The procedure outlined above was followed in the experiments 

 described in this paper. The gelatin was hydrolyzed to various 

 stages by one of the hydrolyzing agents mentioned, pepsin or trypsin 

 then added, and the increase in hydrolysis noted. The reaction 

 was followed by means of a slight modification of the formol titration. 



EXPERIMENTAL. 



Gelatin. — Powdered gelatin, purified by washing at the isoelectric point,* was 

 used in all the experiments. The solutions were made up to contain 5 gm. of 

 gelatin per 100 cc. 



Pepsin. — The pepsin used was an active preparation of Fairchild Bros. (u. 

 s. p. l/ 19,000). It was prepared for use by dissolving 5 gm. in 100 cc. of water, 

 adjusting to pH 2.5 with HCl and dialyzing under pressure against 0.01 n HCl 

 for several days. The solution thus obtained had a very low titration which 

 remained constant. In the concentration used the correction for the pepsin 

 solution was negligible. The solution is referred to as 5 per cent pepsin. 



Trypsin. — Fairchild's trypsin was used. It was prepared for use by dissolving 

 10 gm. in 100 cc. of water and dialyzing under pressure against running tap water 

 at 6°C. for about 18 hours. The solution is very unstable and loses its activity 

 in 3 or 4 days even at 2°C. The formol titration of this solution was negligi- 

 ble in the concentrations used and remained constant. This solution is referred 

 to as 10 per cent dialyzed trypsin. 



Hydrogen ion delerminations. — The determinations were made by the E. M. F. 

 method. 



Formol titration. — The titration was carried out as described in a previous 

 paper.^ The method consists essentialb^ in freeing the solutions from COi by 

 adjusting the pH with acid, and boiling for a few seconds. The solution is then 



♦ Loeb, J., /. Gen. Physiol., 1918-19, i, 237. 



5 Northrop, J. H., /. Gen. Physiol., 1920-21, iii, 715. 



