94 



I. M. KLOTZ 



ing, and the extent of binding is shown here in terms of the free energy, for water 

 alone, for five-tenths molar glycine solution, for one-molar and two-molar solu- 

 tions. As perhaps a first approximation, you might notice that the graph is 

 linear. Perhaps I should have pointed out in the preceding figure (Fig. 1) that if 

 the factor does remain constant, then the free energy should have varied re- 

 ciprocally with the dielectric constant, which to a first approximation it does. 



To a certain extent we have specificity even here in that the only molecules 

 which would effectively increase the dielectric constant by an appreciable 

 amount are the amino acids; but that is a very nebulous specificity. 



I would like to proceed to another example which illustrates the effect of 

 changes in the environment (Fig. 3) in which we have somewhat greater speci- 

 ficity. Here again I will have to give just a little background information. 



We measure the extent of binding in this case and represent it in terms of 

 the following coordinates: the number of molecules of a particular dye (whose 

 structure I shall show you shortly) bound by each protein molecule against 

 concentration of the free dye in the solution, represented here on a logarithmic 

 basis. Then, for the particular protein which is shown here, which is pepsin, 

 if there is no metal present and dye is present, the extent of binding is very 

 small (Fig. 3) and it does not matter whether the pH is anywhere between 4 

 and about 7. I have indicated just two pH ranges in Fig. 3 (by two different 

 symbols), but over the pH range of 4 to 7 there is essentially no binding or 

 very little binding. 



However, if you add to this solution a small amount of zinc, of the order of 

 10~' or 10""* molar, at pH 4 there is a small but definite effect and at pH 5 you 

 can see there is a very substantial effect. The zinc increases the binding (Klotz 

 and Loh Ming, 1954). 



7000 



-AF,* 



6500- 



6000 



.008 



.010 



.012 

 I 



.014 



.016 



Fig. 



DIELECT. CONST. 



2. Free energies of binding of methyl orange by bovine albumin in aqueous 



glycine solutions of different dielectric constant. 



