392 



SCIENCE 



[N. S. Vol. LIII. No. 1373 



solution to form an ionized protein salt. The 

 amount of this salt formed is determined 

 by the hydrogen ion concentration of the 

 solution according to the well-known laws 

 governing the reaction of an acid and a weak 

 base. 



2. The pepsin is present in the solution, 

 (a) as free, probably negatively charged 

 pepsin, and (&) in combination with the 

 products of hydrolysis of the protein. These 

 two forms are in equilibrium with each other 

 and their relative concentration depends on 

 the amount of products of hydrolysis present 

 in the solution as demanded by the law of 

 mass action. 



3. The reaction takes place between the 

 ionized protein and the free pepsin. 



EXPERIMENTAL EVIDENCE FOE THE ABOVE 

 STATEMENTS 



Loeb^ has shown by direct experiment that 

 the protein exists in solution in an equi- 

 librium condition as stated under (1). 



Rekelharing and Einger^ have shown that 

 purified pepsin in solution is negatively 

 charged. It may be shown by direct experi- 

 ment that the addition of products of hydro- 

 lysis decrease the activity of the enzyme and 

 that the amount of the decrease in the 

 activity can be predicted by the law of mass 

 action. 



The validity of the third assumption may 

 best be tested by applying the proposed 

 mechanism to the explanation of the char- 

 acteristic peculiarities of the reaction out- 

 lined under (1 to 5). 



1. Influence of Quantity of Enzyme on the 

 Final Equilibrium. — Since the free enzyme 

 and the products of hydrolysis are in equi- 

 librium there will always be some active 

 (free) enzyme present no matter how high the 

 concentration of products becomes. The re- 

 action will therefore proceed to approximately 

 the same point irrespective of the amount of 

 enzjrme present at the beginning of the re- 



2 Loeb, J., J. Gen. Physiol, 1918-19, 1.; 1919-20, 

 II. 



3 Peckelharing, C. A., and Ringer, W. E., Z. 

 physiol. Chem., 1911, L2XV., 282. 



action. It will be seen, however, that the 

 final equilibrium will depend to a slight ex- 

 tent on the amount of enzyme present since 

 some of the products of hydrolysis are com- 

 bined with the enzyme. 



2. Concentration of Enzyme. — If the en- 

 zyme solution is pure, the rate of hydrolysis, 

 other factors being constant, will be directly 

 proportional to the concentration of enzyme 

 taken. If the enzyme solution contains 

 products of hydrolysis or other substances 

 with which the enzyme is combined then the 

 rate of hydrolysis will increase more slowly 

 than the concentration of enzyme solution 

 since the amount of free enzyme present be- 

 comes relatively smaller the higher the con- 

 centration. 



3. Concentration of Protein. — If the rate of 

 hydrolysis of the protein is proportional to 

 the concentration of ionized protein then the 

 rate must increase more slowly than the total 

 protein concentration since the ionization of 

 the protein salt is less in concentrated than 

 in dilute solution. 



4. Schiltz's Rule. — Ari-henius* has pointed 

 out that in an equilibrium system, such as 

 exists between free pepsin and the products of 

 hydrolysis, the concentration of one of the 

 reacting molecules or ions becomes inversely 

 proportional to the concentration of the 

 second as soon as the second is present in 

 large excess. That is, the amount of free 

 pepsin present, after the first few minutes of 

 the reaction, is inversely proportional to the 

 amount of products formed. It follows from 

 this that the amount of hydrolysis at any 

 time is proportional to the square root of the 

 time elapsed, which is one form of Schiitz's 

 rule. 



5. The Influence of the Hydrogen Ion Con- 

 centration. — It is clear that the more acid 

 is added to the protein the more protein salt 

 will be formed until all the protein is present 

 in the form of protein-acid salt. This salt is 

 practically completely ionized in dilute solu- 

 tion as may be shown by direct measurement 



* Arrheuius, S., ' ' Quantitative Laws in Biolog- 

 ical Chemistry," London, 1915, pp. 36—48. 



