708 14. EFFECTS OF pH ON ENZYME INHIBITION 



fers that are usually included, there will be a significant alteration of the 

 pH. Of course, when possible, the pH of the inhibitor solution should be 

 adjusted previously to the pH of the enzyme preparation, but sometimes 

 this cannot be done because of the insolubility of the inhibitor at the required 

 pH. When the pH's of all the solutions that make up the final test me- 

 dium are not identical, the only recourse is to determine the pH after all 

 additions have been made. However, many reports simply state that a 

 buffer of the designated pH was used and one is left to wonder if the actual 

 pH was checked. 



The pH of a solution of a weak acid or its salt may be estimated easily 

 by the equations: 



Solution of HA: pH = — - [pKa - log (A,)] (14-137) 



Solution of NaA: pH = -~ [-pK^ + pZ„ + log (Aj)] (14-138) 



Z 



where K^, is the ion product of water and (A^) = (HA) -f (A~). Since K^ 

 at 37.50 is approximately 2.5x10"^^, if the measurements are made at 

 physiological temperatures, pE"^, = 13.6. These equations are not exact 

 but are satisfactory for most inhibitors at the concentrations usually used. 

 The presence of a buffer does not by any means ensure a constant pH. 

 It is especially important to know the exact pH at which a reaction is run 

 when the experiment is designed to study the effects of pH variation; it is 

 not enough to set up a series of reaction vessels containing a buffer at 

 different pH's and to assume that these values will not change when any 

 addition is made to start the reaction or an inhiliitor solution added to 

 one vessel of a pair to compare with the uninhibited rate. The buffer ca- 

 pacity of a solution depends on the concentration of buffer and the sepa- 

 ration of the pH from the p/i,, of the buffer. The enzyme solutions thus 

 will not have the same buffering capacity at different pH's and, whereas 

 the addition of an inhibitor at one pH might not alter the pH significantly, 

 at another initial pH the addition would have a marked effect. The pH 

 of a reaction mixture otten changes during the course of the catalysis and 

 the resistance to this change will depend on the initial pH and the buffering 

 capacity. Errors of 0.1 pH unit or less, or changes of this magnitude during 

 the reaction, can produce mistakes in interpretation and lead to the cal- 

 culation of incorrect constants. It is, therefore, essential either to measure 

 the pH after all the components have been mixed or to show by calculation 

 that the buffer capacity is adequate; since there may be several substances 

 involved in the buffer activity in an enzyme preparation, it is usually sim- 

 pler to determine the pH experimentally, both at the beginning and the 

 end of the reaction. 



