REACTIONS WITH PROTEINS 753 



conclusion that no single group can compete very effectively with the SH 

 groups for the mercurials. Indeed, these other groups probably have their 

 orbitals occupied by competing with the various ligands present in the me- 

 dia. However, there are at least three factors which must be taken into 

 consideration. (1) Certain fortuitous arrangements of two or more non-SH 

 groups, perhaps allowing chelation of the mercurial, can increase the affin- 

 ity markedly, as seen in Table 7-4. It is quite possible that occasionally 

 such situations occur on protein surfaces, although generally the opportu- 

 nities for successful chelation must be rare; e.g., the binding to glycylglycine 

 is less than to glycine, and increase in the length of the polypeptide chain 

 wiU probably reduce affinity except in very special cases. (2) The most 

 reactive SH group or groups on a particular protein may not happen to 

 have a strong affinity for a mercurial, due to steric factors or an unfavorable 

 electric field, so that non-SH groups can compete more effectively. Although 

 there is little quantitative evidence, one gets the impression that usually the 

 SH groups of proteins do not bind most mercurials as tightly as do the SH 

 groups of simple thiols, such as cysteine or glutathione, especially since 

 one can often remove a mercurial from a protein quite readily by adding 

 one of these thiols. (3) As pointed out by W. L. Hughes (1950), mercurials 

 will complex with non-SH groups when the SH groups become saturated, 

 or actually before in many cases. Since excess mercurial is often present, 

 especially in enzyme studies, such secondary complexes must be considered, 

 even though the SH groups are reacted first. 



On the experimental side, it has been observed that some proteins bind 

 more Hg++ than corresponds to the SH content and that some of this is 

 relatively weakly bound. Haarmann (1943 a) found that with increase in 

 the pH progressively more Hg++ is bound to various proteins, although 

 only a fraction is really tightly attached to the protein, and postulated that 

 CONH groups might bind Hg"+. More recently, Perkins (1958, 1961) re- 

 ported the binding of 104-130 g-atoms of Hg/10^ g seralbumin and, follow- 

 ing treatment with bromoacetate (blocking SH and amino groups), the 

 binding increased to 190 g-atoms/10^ g protein at pH 5.5. The SH groups 

 could have accounted for only 1 g-atom/10^ g protein and Perkins felt that 

 numerically the only possible binding sites are the C00~ groups. This is 

 a situation in which there is excess Hg++ present for reaction with non-SH 

 groups and, inasmuch as the dissociation constants are not known, it is 

 impossible to compare the affinities for the various groups. Nevertheless, 

 these results conclusively establish the non-SH binding of mercurials under 

 certain conditions, and it would be well to bear this in mind in enzyme 

 inhibition work. 



Examples of Reactions with Specific Proteins 



(A) Ovalbumin. Although not much work has been done with this pro- 

 tein, the results illustrate some of the problems one encounters. Anson 



