246 



rNACTIVATION OF TRYPSIN. U 



and then adding the trypsin and determining the rate of hydrolysis 

 at once, it is necessary to know whether or not the order of mixing 

 the solutions or the time during which the trypsin has been allowed 

 to react with the inhibiting substance has any influence on the result. 

 Table I is a summary of experiments planned to answer this question. 

 It shows that the order of mixing and the time during which the 

 trypsin and inhibiting solution are left together has no effect on the 

 final result. That is, the equilibrium between the trypsin and the 

 inhibitor must be reached practically instantaneously and be quanti- 

 tatively and instantly reversible. (This is only true if the experiment 



TABLE I. 

 Effect of Order of Mixing and Time of Standing. 



Time to change 10 points 



after hours 



at 6°C. 



(1) 25 cc. gelatin + 1 cc. trypsin. 



(2) 25 cc. gelatin + 1 cc. inhibitor + 1 cc. trypsin added after 

 interval shown 



(3) 25 cc. gelatin + 2 cc. mixture of 5 cc. inhibitor + 5 cc. trypsin. 

 (Mixture allowed to stand as noted and 2 cc. then added to 

 gelatin) 



hours. 



hrs. X 102 

 15 

 16 



21 



25 



22 



1.0 hours. 



hrs. X 10> 



16 



21 

 20 



20 



is made under such conditions that the control trypsin solution — 

 without inhibitor^remains unchanged during the course of the experi- 

 ment. If this condition is not fulfilled, the results depend entirely 

 on the length of time and on the temperature at which the trypsin- 

 inhibitor solution has been allowed to stand. )2 



Influence oj the Gelatin Concentration. 



The retardation could be qualitatively accounted for by assuming 

 that the inhibiting substance combines with the gelatin instead of 



2 Northrop, J. H., J. Gen. Physiol, 1921-22, iv, 261. 



