JOHN H. NORTHROP 61 



is doubled. Since in completely hydrolyzed gelatin the free carboxyl 

 groups (or the amino nitrogen) is increased about twenty times, 

 the hydrolysis due to pepsin alone corresponds to about 5 per cent 

 of the total hydrolysis. It will be seen from the figure that this 

 result is independent of the amount of pepsin added and also of the 

 stage of hydrolysis at which the pepsin is added. It is not possible 

 to say, however, whether or not this really represents the complete 

 action of the pepsin, since the hydrolysis is still continuing slowly 

 and if the analyses were made at weekly intervals it would be found 

 that the values were increasing. This slow increase, however, ap- 

 proaches asymptotically the increase due to the acid alone, so that it is 

 impossible to say when the action of the pepsin stops. The real end- 

 point for pepsin digestion could be definitely fixed only by reaching the 

 same value from both sides. For the purpose of these experiments 

 it is permissible to take the end-point as that point at which the 

 addition of more pepsin causes no further hydrolysis in the course 

 of 3 or 4 days (the action of the acid alone in this time is within the 

 limits of error of the method). In the experiment just described this 

 value evidently corresponds to a titration of 4 cc. of 0.1 n NaOH per 

 10 cc. of 5 per cent gelatin. 



Action of Pepsin on Gelatin Partially Hydrolyzed by Trypsin. 



The results of this experiment are shown in Fig. 2. The experiment 

 shows that the increased hydrolysis due to the pepsin becomes less the 

 farther the hydrolysis due to the trypsin has been carried. The 

 linkages which are split by pepsin are also evidently attacked by 

 trypsin. At the same time, however, the trypsin is also hydrolyzing 

 some linkages which are not attacked by pepsin. This is shown by 

 the fact (Table I) that the addition of pepsin to a solution which has 

 been hydrolyzed by trypsin to a titration of 7.0 cc. causes a still 

 further increase, although, as we have seen, pepsin alone can only 

 carry the hydrolysis to 4.0 cc. The same result was obtained by 

 Henriques and Gjaldbak^ with other proteins. 



