114 COMBINATION OF ENZYME AND SUBSTRATE 



Bayliss^ attaches considerable importance to the combination of 

 enzyme and substrate and considers it an essential point in the theory 

 of enzyme action. Van Slyke and Cullen^ were able to formulate 

 the kinetics of enzyme action on the basis of the law of mass action 

 by assuming the existence of a compound between enzyme and 

 substrate. 



The study of this combination, however, has not furnished any 

 quantitative experimental data as to the nature of the reaction or 

 the influence of various factors on it — due largely to the difficulty 

 of determining quantitatively the amount of enzyme. 



It seemed important, therefore, to obtain quantitative experi- 

 mental data on this subject. In order to do this it was necessary 

 to have a convenient and accurate method for the determination 

 of pepsin. It was found that the change in the conductivity during 

 the digestion of egg albumin by pepsin afforded such a method, 

 Sjoqvist^ found that there were marked changes in the conductivity 

 during pepsin digestion. His results were confirmed with the ex- 

 ception that the change was found not to follow the actual rate of 

 digestion. It can be used therefore only as an empirical method 

 for the determination of pepsin and not for the study of the kinetics 

 of the reaction, as was done by Sjoqvist. The reason for this diver- 

 gence is probably due to the fact that the change in conductivity 

 is due to two causes; (1) the Hberation of free acid (carboxyl) groups 

 which increase the conductivity, and (2) the liberation of free amino 

 groups which bind some of the free acid and so decrease the conductiv- 

 ity. This explanation is borne out by the following facts. 



With dilute solutions of egg albumin containing strong acetic acid 

 (pH 2.3) there is a regular small increase in the conductivity which 

 nearly parallels the increase in free amino groups as followed by the 

 increase in amino nitrogen. This is due to the fact that, owing to 

 the very large excess of free undissociated acid present, the ions 

 which are removed from solution by combination with the free amino 

 groups are replaced by the dissociation of more acid, and so kept 

 nearly constant. The slight increase in conductivity is therefore 

 due to the hberation of free carboxyl groups. In strong acid solu- 



« Van Slyke, D. D., and CuUen, G. E., /. Biol. Chcm., 1914, xix, 141. 

 ' Sjoqvist, J., Skandin. Arch. Physiol, 1893-95, v, 277. 



