638 4. SULFHYDRYL REAGENTS 



The reactions of most SH reagents with thiols depend on the pH and this 

 undoubtedly relates to the ionizations of both SH reagent and the SH groups 

 reacted. In most cases, as with the mercaptide-forming reagents and the al- 

 kylating agents, the rate and degree of reaction increase with increasing 

 pH, and it is likely in these cases that the ionized mercapto anion, R — S", 

 is more reactive than the un-ionized R — SH form. One may visualize some 

 of these reactions as a competition between the SH reagent and a proton 

 for the R — S~ group. The reaction with a heavy metal ion, for example, 

 may be written as: 



R— SH + Me+ ^ R— S— Me + H+ 



and it is obvious that increase of the pH will favor the formation of the 

 mercaptide complex, or, to put it in another way, that the reaction: 



R— S- + Me+ ^ R— S— Me 



will proceed more readily. On the other hand, reactions with double bonds 

 (as with maleate or quinones) or oxidations to the disulfide may occur more 

 readily when the SH group is un-ionized. In any event, the state of ioniza- 

 tion of the SH group is important in enzyme inhibition and may account 

 partly for the different reactivities of protein SH groups. The ionization of 

 SH groups is well discussed by Edsall and Wyman (1958), mainly on the 

 basis of work by Benesch and Benesch (1955). The ionization microcon- 

 stants for cysteine are given in Table 1-14-4. It is clear that the pK^ of an 

 SH group is markedly dependent on the electric field present, that is, on 

 the vicinal ionic groups. One might roughly estimate the p^,/s for an SH 

 group as shown in the following tabulation: 



pK„ 



Near a + charged group 7.2-8.5 



No electric field 8.5-9.2 



Near a — charged group 9.2-10.2 



On the surface of a protein the electric field will be the resultant of all the 

 contributions of the ionic groups. Enzyme SH groups must therefore exhibit 

 a wide range of ionizations at any designated pH, and in most cases will 

 exist mainly in the un-ionized form at physiological pH. The piii^ of the SH 

 groups on aldolase in 4 M urea is around 8.66, but the native enzyme has 

 6 exposed SH groups with \)K„ values near 10.5 and buried SH groups with 

 an apparent p^„ of 11.5 (Donovan, 1964). 



