INTERACTIONS OF SMALL MOLECULES WITH PROTEINS 315 



The Zn++ and Cu++ thus behave differently: the binding of 4-methylimi- 

 dazole to Cu++ decreases the affinity for succeeding molecules, whereas 

 binding to Zn++ increases the affinity. The explanation probably lies in 

 the planar configuration of the Cu-N bonds and the tetrahedral arrangement 

 of the Zn-N bonds. The evaluation of successive constants was first treated 

 thoroughly by Bjerrum (1941) and a very comprehensive presentation is 

 given by Edsall and Wyman (1958, p. 591). 



Similar interactions between binding sites may well occur on enzymes 

 and in such cases the determined K^ or K^ may not relate to any specific 

 binding but represent only the mean value over the range studied. For 

 'positive interactions (where the binding of a molecule helps the binding of 

 another molecule) the determined K^ might be smaller than for the uncom- 

 plicated binding of the first inhibitor molecule, whereas for negative inter- 

 actions (where the binding of a molecule hinders further binding) it would 

 be larger. 



It is probable that is some cases the total number of binding sites for 

 an inhibitor is greater than the number of enzymically active sites; that 

 is, the inhibitors may be bound to both active and inactive sites. Binding 

 to inactive sites would not necessarily affect the active sites with respect 

 to enzyme rate but might affect the binding of other inhibitor molecules 

 to the active sites and thus modify the determined K^. Although this effect 

 is not generally very significant, it may well be important in mutual de- 

 pletion systems (pseudoirreversible or titration inhibition), inasmuch as 

 the characteristic behavior of such systems depends on the reduction in 

 inhibitor concentration, and it is of no importance where the inhibitor is 

 bound. It is very difficult, for example, to estimate the number of active 

 sites from such titration inhibition data, even with pure enzymes, due to 

 possible binding with other groups. This problem has been referred to in 

 Chapter 3 and will be taken up in more detail in Chapter 15. 



Effect of Total Protein Charge on Binding 



Except at their isoelectric points, proteins possess a net charge and it 

 has been frequently assumed that this net charge determines whether ions 

 will be bound or not. However, there are many, instances where ions are 

 bound to proteins although the signs of the charges are the same. Mg"^"*" 

 is bound to lysozyme at pH 7.4, although at this pH the protein is posi- 

 tively charged (Carr and Woods, 1955) and the anion of methyl orange is 

 bound to serum albumin throughout a pH range above the isoelectric point 

 when the protein is negatively charged. Undoubtedly a net protein charge 

 must influence many interactions electrostatically, and probably prevents 

 some from occurring, but it is not necessarily the deciding factor. Presum- 

 ably on a protein that has a net negative charge, there are regions where a 



