506 KINETICS OF TRYPSIN DIGESTION 



Rate of Hydrolysis as Measured Directly by the Disappearance of the 



Substrate. 



It has been shown above that the rate of formation of the products 

 of hydrolysis of gelatin or casein by trypsin does not increase in pro- 

 portion to the concentration of substrate but increases much more 

 slowly and becomes independent of the substrate concentration when 

 the latter is more than 2 or 3 per cent. It was also shown that this 

 peculiarity could not be accounted for by assuming the existence 

 of an inter-^ediate compound between the enzyme and substrate nor 

 by the assumption that the hydrolysis was proportional to the ionized 

 protein. In these experiments as in most experiments with enzymes 

 the hydrolysis was followed by determining the amount of the prod- 

 ucts formed and assuming that the amount of substrate remaining 

 is the difference between the amount of products found at any time 

 and the total amount that can be formed under the most favorable 

 conditions. It is well known that trypsin digestion consists of a 

 series of consecutive reactions since a number of products may be 

 isolated from a digestion mixture which can still be acted on by the 

 enzyme. It seemed possible therefore that the peculiar results 

 discussed above were due to the fact that the increase in the products 

 of reaction does not correctly represent the decrease in the substrate 

 concentration. It is the change in concentration of the latter value 

 that is predicted by the law of mass action. An experiment was 

 therefore performed in which the digestion was followed by determining 

 the increase in amino nitrogen and also the decrease in unchanged 

 casein. The results are given in tables XI and XII. The tables 

 show that the two methods give entirely different results. As meas- 

 ured by the increase in amino nitrogen the rate of hydrolysis is practi- 

 cally independent of the casein concentration, whereas when the 

 change in the casein concentration is measured directly the rate of 

 digestion is very nearly proportional to the concentration of casein 

 as demanded by the law of mass action. The constant calculated 

 from the monomolecular formula still shows a drop with increasing 

 hydrolysis. This is more marked in the concentrated than in the 

 dilute solution and is the result expected owing to the inhibiting action 

 of the products of hydrolysis. When the rate of hydrolysis is de- 



