500 KINETICS OF TRYPSIN DIGESTION 



Effect of Varying the Amount of Trypsin or Inhibiting Substance. 



It has been shown above that in order to account for the fact that 

 the percentage retardation is independent of the substrate concen- 

 tration, it is necessary to assume that the inhibitor-trypsin compound 

 is only slightly dissociated. This assumption, as has already been 

 pointed out, is contradicted by the experiments in which the amount 

 of trypsin or inhibitor is varied. This is shown in Tables VII and 

 VIII. In these tables the results under Calculated I were obtained 

 from the equation used to calculate the results in Table VI, and which 

 is derived by means of the assumption that the inhibitor-trypsin 

 compound is only slightly dissociated. The table shows that the 

 formula will not serve even as a first approximation in spite of the 

 fact that it contains three arbitrary constants. The figures given 

 under Calculated II were obtained by aid of the assumption that the 

 enzyme-inhibitor compound is widely dissociated and that the rate 

 of hydrolysis is proportional to the free enzyme.^^ They agree well 

 with the experimental values. 



These experiments show that the results obtained when the gelatin, 

 inhibitor, and tr5^sin concentrations are all varied cannot be ac- 

 counted for on the assumption that the tr3rpsin becomes saturated 

 with substrate. They seem to be conclusive even though it is assumed 

 that the equilibria are not governed by the law of mass action, since 

 in order to explain one set of experiments (gelatin constant, trypsin 

 or inhibitor varied) it is necessary to suppose that the trypsin-inhibi- 

 tor compound is widely dissociated while in the other set of experi- 

 ments (trypsin and inhibitor constant, gelatin varied) it is necessary 

 to suppose that the same compound is very slightly dissociated. 

 This is true irrespective of the quantitative law that is assumed to 

 govern the equilibrium. There is much more direct experimental 

 evidence in favor of the trypsin-inhibitor compound than of the 

 ' trypsin-gelatin compound. 



There is no doubt on the other hand that the rate of hydrolysis 

 does not increase in proportion to the gelatin concentration as ex- 

 pressed in grams per Kter. If it is assumed then that the reaction is 



^^ For the derivation of this equation see Northrop, J. H., /. Gen. Physiol., 

 1921-22, iv, 230. 



