170 J. F. PECHÈRE AND HANS NEURATH [10 



the same rate law, indicating that they are all manifestations of one and the 

 same molecular event. The correlation between activity and amount of iso- 

 lated hexapeptide indicates that the lysyl-isoleucine bond is broken simul- 

 taneously with activation. The correlation between activity and total amount 

 of trypsin appearing indicates that no intermediate compound having 

 enzymatic activity is formed during the activation process, a conclusion 



'0 20 40 60 80 

 Time (min.) 



20 40 60 80 

 Time (min) 



Time (min.) 



ACTIVATION 



OF 



■ ' 20 ' 40 60 80 

 Time (min.) 

 TRYPSINOGEN 



Fig. 1. Correlation between experimental parameters and appearance of enzymatic 

 activity during the autocatalytic activation of trypsinogen. The following conditions 

 applied to all the experiments: trypsinogen concentration approx. 12-5 mg/ml.; trypsino- 

 gen/trypsin ratio 20 : 1 ; 0-1 M Tris buffer (pH 8 0) containing 0-05 M-CaCl2; temperature 

 approx. 0° C. In all experiments except those shown on the left top quadrant, indole was 

 also present to minimize the action of a slight chymotryptic impurity which was found 

 to be present in the trypsinogen and trypsin preparations used. 



which clearly differentiates the activation of trypsinogen from that of chymo- 

 trypsinogen.^ The correlation between activity and change in optical rota- 

 tion supports the view that molecular rearrangement (toward a more nearly 

 helical configuration) is an essential aspect of the activation process; and 

 the correlation between enzymatic activity and base consumption is in 

 accord with the assumption that one and only one peptide bond is broken 

 during the reaction. This latter conclusion is dependent in part on an accu- 

 rate knowledge of the pK of the amino group which is formed during 

 enzymatic hydrolysis. In the present experiments, a pK of 81 at 0° was 

 assumed, whereas a pK of 7-6 would have to be assumed if two bonds were 

 broken under these conditions. It is also recognized that the hydrolysis of 



