314 6. INTERACTIONS OF INHIBITORS WITH ENZYMES 



tinent now to point out that the binding of the dye methyl orange to se- 

 rum albumin has been studied quantitatively in this regard (Klotz, 1949). 

 The buffer anion can compete with the dye anion for binding sites and the 

 effect is thus dependent upon the concentration of the buffer anion. Even 

 the CF ion can interfere with the dye binding by this mechanism. 



Multiple Binding Sites on One Protein Molecule 



In the expressions of enzyme kinetics, (E) refers to the concentration 

 of active sites, not necessarily enzyme molecules, and hence the number 

 of sites on a single enzyme molecule is of no consequence in these formu- 

 lations unless the sites can interact with one another. Interactions between 

 sites on enzymes have not been adequately studied but situations analogous 

 to hemoglobin, where the four heme sites are interdependent, may well 

 occur. The kinetics of multiple interactive sites have been presented (Chap- 

 ter 3) and the forces responsible for such interaction may now be attributed 

 to a direct influence (electrostatic or steric) or an indirect influence mediated 

 through the protein. In the binding of small ions or molecules to proteins, 

 it has been found that several sites are usually present on a molecule. For 

 serum albumin the following number of sites have been demonstrated: 

 Cu++, 16; Ca++ and Mg++, 8; dodecyl sulfate, 14; phenylbutyrate, 24; 

 methyl orange, 22; and testosterone, 9. Interactions between such sites 

 have been found. 



The binding of Cu"'""'" to serum albumin at 25° is characterized by a re- 

 duction in binding energy with each Cu"^"^ ion bound. The first Cu"^"^ ion 

 is bound with JF = — 5.91 kcal/mole whereas the last or sixteenth Cu++ 

 ion is bound with JF = — 1.72 kcal/mole, a reduction of 0.26 kcal/mole 

 for each Cu++ bound on the average. This effect is due to two factors: the 

 statistical effect, whereby the progressive occupancy of sites restricts the 

 probability for a Cu++ ion finding an unoccupied site, and an electrostatic 

 effect resulting from the altered electrical field of the protein upon binding 

 of the Cu"^"^ ions (Klotz, 1949). Simpler systems are presented by the com- 

 plexes of metal ions with nitrogenous substances, such as Zn(NH3)4"'"'^, 

 Ni(NH3)e"'""'", and Cu(imidazole)4"^"^, where the binding of each successive 

 ammonia or imidazole is altered by the previously bound molecules. The 

 complexes of 4-methylimidazole have been studied recently by Nozaki 

 et at. (1957) and the successive p£''s (the K is here the intrinsic dissociation 

 constant in order to eliminate the statistical factor) were found to be: 





