98 



I. M. KLOTZ 



8 



PH 



Fig. 7. Effect of pH on binding constant 



To prove to ourselves that this could account almost entirely, within experi- 

 mental accuracy, for the increase, we calculated, as is shown in the next illus- 

 tration (Fig. 7), the actual binding constant (Hughes and Klotz, 1956) for the 

 process of dye going on to the zinc which is already attached to the protein. 

 In calculating this association constant, which is plotted in Fig. 7, we have 

 already taken into account in our equation the extra zinc which is bound by 

 the protein. With this correction the constant is substantially level over the 

 pH range of 6-7.5. In other words, you can account entirely for the increase 

 in the extent of binding in terms of the extra zinc which is present on the pro- 

 tein. 



However, just as before in the photometric curve (Fig. 5), there is a very 

 steep drop-off as soon as you exceed about pH 7.5, either in the constant (Fig. 

 7) or in the extent of binding measured semi-quantitatively (Fig. 5). This was 

 a puzzle but only for a short time. 



The explanation must lie, as is shown in the next figure (Fig. 8), in hydrolytic 

 changes. I would like to point primarily to the first equation; I shall come back 

 to the others in a moment. The explanation must lie in the hydrolytic equilib- 

 rium of the zinc. We have to keep in mind that OH ions at increasing pH's 

 become of importance and can displace the dye from the metal protein combi- 

 nation by a displacement or substitution mechanism. 



If this is true, then we would expect that not only should this mechanism 

 work for zinc, but since we have studied 8 or 10 other metals, it should work 

 for these other metals also. Since the affinity for hydroxyl ion of other metals 



