VARIATION OF ENZYME INHIBITION WITH pH 709 



The buffer value of van Slyke is defined as dXld\)]l where X stands for 

 the moles of strong acid or base added per liter of solution. Its value is 

 given by: 



dXfdvB. = 2.3a(l - a){B,) (U-139) 



where a = (B-)/(B^) and (B^) is the total concentration of buffer. Ali)ha 

 is also given by K^IKR^) -t- K^]. The maximal buffer value is exhibited 

 when pH = p^^ and a = 0.5, at which point f/Z/f^pH = 0.575(B,), and 

 it falls off on either side. Let us consider a 0.02 M phosphate buffer with 

 pK^ = 7.2. If the pH of the solution is 7, a = 0.389 and flZ/c^pH = 0.011. 

 This means that rfpH/f?Z = 90 so that the addition of a 5 mM strong 

 acid will shift the pH 0.45 units; actually, the shift will be somewhat greater 

 because as the pH is lowered, the buffer value decreases. If the pH had 

 been 6, a = 0.0593 and dXIdvR = 0.0017, so that dpRjdX = 590 and 

 addition of 5 mM strong acid would shift the pH at least 3 units. The 

 addition of a weak acid will, of course, have less effect, but can be appre- 

 ciable; for example, the addition of 10 mM malonic acid to a 0.02 M phos- 

 phate buffer at pH 7 will lower the pH to 4.7. On the other hand, the addi- 

 tion of 10 mil/ sodium malonate will affect the pH negligibly. As a general 

 rule, when the pH is initially one unit or more from the pZ^ of the buffer, 

 the buffering capacity will usually be low and care must be taken to prevent 

 or detect changes in the pH. 



Buffer ions occasionally interact with the enzyme or other components 

 of the system, and a change in pH — by modifying the concentrations of 

 these buffer ions — may exert effects on the binding of the substrate or the 

 inhibitor. We have observed that the i:>KJs of the two enzyme groups on 

 fumarase are different in acetate or phosphate buffers (Table 14-3). Alberty 

 (1954) has examined the rates and constants of the fumarase reaction 

 for their dependence on the concentration and pH of buffers, and has 

 developed a theory of buffer effects that applies to inhibition. The var- 

 iation of the apparent inhibitor constant with pH and concentration of 

 buffer is represented by an equation of the type: 



where: 



' /«./' 1 +L.(B) 



ft, f n 



LJhhe jr I Jheb j-r 



1 = —? — ;^ ^be "T r ,, ""- e 

 J he J he 



Jhbeb 



L. = 



Jhe J^be-^beb 



Jhbei -^ei 

 Jhei "-be^bei 



