490 KINETICS OF TRYPSIN DIGESTION 



Experimental Methods. 



The rate of hydrolysis was followed by means of the change in 

 conductivity of the solution as already described.^ The experiments 

 were all conducted at a pH of 6.0. 



Trypsin. — The trypsin was a sample of Fairchild's trypsin and 

 was purified for use by dialysis under pressure. 



Cooper's gelatin was used and was rendered ash-free by washing 

 at the isoelectric point as described by Loeb.^" The inhibiting solu- 

 tion was made by allowing trj^sin to completely digest gelatin 

 and then concentrating the solution in vacuo. 



Method of Measuring the Rate of Hydrolysis. — In order to obtain a 

 correct measure of the rate of hydrolysis it is necessary to compare 

 the reactions at the same stage. The rate of digestion decreases 

 rapidly with the progress of digestion for two reasons: first, the con- 

 centration of substrate is decreasing; second, the concentration of 

 active enzyme is decreasing owing to the inhibiting action of the 

 products of digestion. If the reactions are compared at a point of 

 equal percentage hydrolysis, the change in substrate concentration 

 is corrected for but the change in enzyme concentration will be 

 very different. The small amount of enzyme will be inhibited to a 

 larger extent than the large amount. If the reactions are compared 

 after equal times, both conditions are varied. If, however, the time 

 to cause a very small amount of hydrolysis is taken, the change in 

 substrate concentration may be considered negligible and the effect 

 on the enzyme will be small and nearly the same in both cases. 

 This method, therefore, gives the most significant value. 



The result of an experiment with 1 and 5 per cent gelatin and 1 

 and 10 units of trypsin is shown in Figs. 1,2, and 3, in which the in- 

 crease in specific conductivity of the solution has been plotted against 

 the time in hours. Table I gives the time required to cause an equal 

 percentage of the total change in the two gelatin concentrations with 

 the different enzyme concentrations. The table shows that the time 

 required for the hydrolysis to be completed to any given percentage 

 in the two solutions, is not the same (as would be predicted by the 

 monomolecular formula), but is very much greater for the 5 than for 



10 Loeb, J , /. Gen. Physiol, 1918-19, i, 237. 



