JOHN H, NORTHROP 601 



the consequent slowing up of the enzyme due to the inhibiting effect of the prod- 

 ucts. It is also clear that the retardation will be proportionally greater if a 

 small amount of enzyme is present than if a large amount is present (irrespective 

 of the mechanism by which the retardation is affected). The same reasoning 

 holds for the case when the amount of products formed in a given time is taken 

 as the measure of the rate of reaction. This question was discussed fully in a 

 previous paper.^* 



In all the experiments given in this paper, therefore, the rate of digestion is 

 measured as the reciprocal of the time necessary to cause a small absolute change 

 in the substrate concentration. According to the law of mass action as applied to 

 monomolecular reactions the time necessary to cause this change should be nearly 

 inversely proportional to the substrate concentration, provided the change is 

 small compared to the total change in the lowest concentration. If wider varia- 

 tions than this are used it is necessary to calculate the predicted time according 

 to the monomolecular formula. It may appear that the above method of testing 

 the reaction is a very indirect one and that a simpler and more exact method would 

 be to express the course of a single reaction, according to the mechanism proposed, 

 in a single equation. This equation could then be tested experimentally. Such 

 a procedure, however, leads inevitably to an equation with two or more constants, 

 the value of which must be determined from the experiments themselves, so that 

 but little weight can be attached to the agreement of such an equation with the 

 experimental facts. It seems better, therefore, to limit the experimental condi- 

 tions in such a way as to leave but one variable. 



In all the experiments reported in this paper, the changes are within the above 

 limits and the time required to cause a constant change should, therefore, (ac- 

 cording to the mass law) be nearly inversely proportional to the substrate concen- 

 tration at the beginning of the reaction. As will be seen this is not the case if 

 the total concentration of protein is considered as the active concentration but 

 is approximately true if the concentration of ionized protein is considered as the 

 reacting mass. 



In these experiments the rate of hydrolysis was followed by means of changes 

 in the conductivity of the solution. It is, therefore, necessary to be sure that the 

 production of the same amount of peptone in each of the solutions used causes the 

 same change in conductivity. This was tested experimentally in each experi- 

 ment by adding 1 cc. of peptone solution (prepared from egg albumin by the ac- 

 tion of pepsin) to 25 cc. of the protein solution and determining the change in 

 conductivity. It was found that the addition of an equal amount of peptone to 

 protein solutions of varying concentrations (from 20 to 1 per cent) does cause an 

 equal change in conductivity provided the hydrogen ion concentration of the solu- 

 tion is greater than pH 1.8. If the solution is less acid than this the change in. 

 conductivity of the solution on the addition of a constant quantity of peptone in 

 the presence of a large amount of protein is less than that caused in the presence 

 of a small amount of protein. This is obviously due to the buffer action of the 

 protein in high concentration and can be foreseen from the titration curve of the 

 protein. 



