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14. EFFECTS OF pH ON ENZYME INHIBITION 



to 80 in this range. Below a pH^ of 4, d^HldX would again decrease due to 

 the carboxylic groups on the proteins. It may well be that some of these 

 values of dpHjdX, especially for phosphate, are too low, since all of the 

 ionizing groups may not be free. It is not improbable that when cells are 

 exposed to an acid solution of a 10 niM weak acid, between 100 and 200 

 raM HA can enter the cell and release the equivalent amount of H"*". This 

 would drop the pH, by at least 4 units if the mean dpH/dX in this 

 range were 40. 



Plotting of Equieffective Concentrations of Inhibitor Against pH 

 (Simon-Beevers Curves) 



Now that certain factors that may play a role in the pH dependence of 

 intracellular inhibition have been discussed, we may return to the original 

 attempt to formulate these phenomena in a quantitative fashion. Some in- 

 formation may be obtained from plotting the total external inhibitor con- 

 centration required to produce a standard chosen depression against the 

 external pH^. Although this method of plotting was not originated by Si- 

 mon and Bee vers, they applied it generally to the actions of weak acids 

 and bases and have more than anyone developed the theoretical implica- 

 tions (Simon and Beevers, 1952). 



Simon and Beevers pointed out that usually the most easily determined 

 effect is a 50% response in the cells. With regard to inhibitors this means 

 a 50% depression of the reaction or event measured. Thus one may apply 

 this to individual enzyme effects or to a complex metabolic process or 

 to some cellular activity, such as contraction or growth. It is very impor- 

 tant to determine the equieffective concentrations accurately. For this pur- 

 pose it is necessary to use several concentrations of the inhibitor that pro- 

 duce effects both less and greater than the 50% reduction at each pH^, 

 and to plot the data to determine the intersection of the curve with the line 

 representing 50% reduction. It is also necessary to make certain that the 

 pHo for each concentration of the inhibitor is constant by determining it 

 directly. 



We shall assume that the inhibitor is a weak acid and that the active 

 form within the cell is the anion, I. It must also be assumed for the present 

 that any changes in pH^ will not alter the inhibition produced by I. Equa- 

 tion 14-153 for the completely buffered case can be rewritten as: 



(I.). = (I). 



(H), 

 (H)„ 



1 + 



(H), 



K„ 



Taking the logarithm of both sides: 



log (!,)„ = log (I), - pH, + pH„ + log 



(H), 



K„ 



(14-161) 



(14-162) 



