VARIATION OF INTRACELLULAR INHIBITION WITH pH 735 



tion of inhibitor were derived. The problem of an unknown buffer capacity 

 can sometimes be circumvented when potent inhibitors are used. If a sub- 

 stance is a very active inhibitor, the concentration in the medium need 

 not be high to produce satisfactory inhibition and hence only a small 

 amount of the uncharged form will penetrate into the cell. In such cases, 

 the pH, would be changed very little even though the buffering was only 

 partial. It may, therefore, be assumed that the cells are completely buffered 

 when sufficiently potent inhibitors are used. 



What is the maximum concentration of inhibitor that can be used without 

 changing the internal pH, significantly? Let us require that pH, change 

 not more than 0.2 pH unit and that dpHldX = 15 near the normal pH^ of 

 the cell. Less than 13 mM HI must enter the cell on this basis. Since in 

 the low pHp range the maximal concentration of H+ released by HI within 

 the cell is nearly sixty-five times the external inhibitor concentration, it 

 follows that the maximal allowable inhibitor concentration in the medium 

 is 0.2 mM. Let us then define a potent inhibitor in this connection as one 

 requiring less than 0.1 mM concentration to produce appreciable inhibition 

 within cells, providing a margin of safety for those cells whose buffer 

 capacity may be less than the assumed value. Since around neutrality, 

 (I), is approximately equivalent to (I,),, (see Fig. 14-16), the inhibition must 

 be characterized by a K, of less than 10~^ M. On the other hand, a very 

 weak inhibitor for which the external concentration must be 10 mM or 

 over can be treated as conforming roughly to the unbuffered situation, 

 especially in the low pH^ range. 



Relation between the Inflections of Inhibition Curves and the pK^ 



There are two common ways of plotting the pH dependence of cellular 

 inhibition: the direct plot of the degree of inhibition against pH^ (the 

 t'-pH^ plot) and the plot of equieffective inhibitor concentrations against 

 pH^ [the log (I^)o-pHq plot]. The inflections of the latter curves will always 

 occur at pH^, values corresi^onding to the pK^'s of the inhibitors. However, 

 this is not necessarily true for the ^-pH^ type of plot. It has occasionally 

 been assumed by investigators that the midpoint of an i-pHo curve should 

 give the pK^ of the inhibitor and some concern has been expressed when 

 the inflection does not coincide with a known p^^. The pH^ at which the 

 midpoint of the inflection occurs is actually dependent on the concentra- 

 tion of the inhibitor in the medium. Inhibition is plotted against pH^ 

 in Fig. 14-25 for a potent inhibitor so that variations in pH, can be neglected. 

 It is seen that the inflection midpoints are shifted to higher pH^ values 

 when the inhibitor concentration is raised. The inflection occurs near the 

 pK^ only at the lowest concentrations of inhibitor. 



