JOHN H. NORTHROP 229 



way with the enzyme. Bayliss considered that the amino-acids were 

 probably responsible for this inhibiting effect. It would seem, there- 

 fore, that these substances are favorable for the purpose of the present 

 experiments since they are also involved in the kinetics of the reaction, 

 and are chemically well defined substances. It was found, however, 

 that the amino-acids themselves have very little if any effect, if the 

 effect on the pH was controlled, whereas the solution resulting from 

 the hydrolysis of the protein by trypsin was strongly inhibitory. 

 Such solutions were, therefore, used although it was not found pos- 

 sible to determine exactly what chemical compound was responsible 

 for their reaction. 



Methods Used in the Present Investigation. 



Inasmuch as the quantity of an enzyme can only be determined by measuring 

 the rate at which it reacts it is necessary to have some convenient and accurate 

 method for following the course of the hydrolj^sis. In the present experiments 

 it is also necessary to arrange conditions in such a way as to prevent any change 

 in pH during the course of the hydrolysis. Henri and des Bancels^ found that 

 the hydrolysis of proteins by trypsin could be accurately followed by noting the 

 change in the conductivity of the solution. The same method was used by 

 Bayliss who also showed that the change in conductivity was not caused by 

 changes in the viscosity of the solution and was parallel to the amount of non- 

 precipitable nitrogen formed. A number of experiments were made to determine 

 the best conditions to use for the purpose of the present experiments. It was 

 found that at a pH of 6.2 to 6.4 no change occurred in the pH during the hydrolysis 

 of gelatin (due probably to the fact that this is near the isoelectric point of the 

 products formed), so that it was not necessary to use buffer solutions if the deter- 

 minations were made in this range. This is a great advantage since buffers 

 interfere with the determination of the conductivity. Gelatin was found to be 

 the best protein since it gives clear solutions and is easily prepared in the necessary 

 salt-free condition. Since trypsin is very unstable in solution the experiments 

 were made at 33°C. The change in the conductivity of the solutions was fol- 

 lowed by the change in the readings of the Kohlrausch bridge. These readings 

 are proportional to the percentage change in the conductivity, so that the lower 

 the original conductivity of the solution the greater will be the change in the 

 bridge readings for a given absolute increase in the conductivity of the solution. 

 On the other hand, the lower the original conductivity the more sensitive the 

 solution is to the addition of traces of salts, to temperature change, etc. It was 

 found better, therefore, to increase the conductivity of the gelatin by the addition 

 of KCl until the solution had a specific conductivity at 33°C. of 2 X 10"^ reciprocal 

 ohms. 



8 Henri, V., and des Bancels, L., Compt. rend. Biol, 1903, Iv, 563, 787, 788. 



