498 KINETICS OF TRYPSIN DIGESTION 



The direct experimental evidence shows conclusively that the en- 

 zyme and the inhibiting substances are combined to form a highly 

 dissociated compound, even though the agreement of the experi- 

 ments with the results calculated from the law of mass action be con- 

 sidered to be accidental. If the substrate is also combined with the 

 enzyme it should be possible, by increasing the substrate concentra- 

 tion sufficiently, to cause all the enzyme to combine with the sub- 

 strate. In other words, the higher the substrate concentration the 

 less should be the effect of the inhibiting substance. This may be 

 seen from the following equation: 



+ gelatin ;:± trypsin-gelatin 



trypsin cr: ► 



+ inhibitor ;:^ trypsin-inhibitor 



Increasing the concentration of gelatin will cause the equilibrium to 

 be shifted in the direction of the large arrow; i.e.; it will cause the 

 amount of trypsin combined with the inhibitor to become less. This 

 will be true even though the equilibrium is not one which follows the 

 law of mass action, as long as the equilibrium is reversible and the 

 trypsin-inhibitor compound widely dissociated. It has already been 

 found that the trypsin-inhibitor compound is readily dissociated 

 irrespective of any assumption as to the nature of the compound. 

 Table VI is a summary of experiments made with constant amounts 

 of trypsin and inhibiting substance and increasing concentrations 

 of gelatin. The figures are the averages of four to six determinations. 

 The table shows that the retardation due to the inhibiting substance 

 is independent of the gelatin concentration. In order to account 

 for this result, if the velocity of hydrolysis depends on a trypsin-gela- 

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

 compound is only very slightly dissociated, and that the inhibiting 

 substance and trypsin are present in about the same concentration. 

 The figures under Calculated I were obtained by means of the law of 

 mass action based on these assumptions. They approximate the 

 experimental values and agree with the experimental result that the 

 percentage retardation is independent of the gelatin concentration. 

 If it be assumed, as was done by Michaehs and Menten, that the 

 inhibiting substance is present in very much higher concentration 

 than the enzyme, the figures given under Calculated II are obtained. 

 They are evidently incompatible with the experiment. 



