644 4. SULFHYDRYL REAGENTS • 



ious SH reagents, and (4) the lack of correlation between the reactivity of 

 SH groups and their relationship functionally or spatially to the active 

 center. These principles are central to the problem of inhibition by SH re- 

 agents and require some general discussion relative to the possible mecha- 

 nisms involved. 



The various, and mostly obvious, hypotheses to explain the differential 

 reactivity of SH groups have frequently been presented with a prolixity 

 inversely proportional to the amount of evidence available. Indeed, at the 

 present time there is little, if any, positive evidence for any explanation, 

 but there are a number of factors that must be of some importance, and 

 these can be enumerated. It should be emphasized that differential reactivity 

 should be based on accurate spectrophotometric or argentimetric titrations 

 of the enzyme SH groups under various conditions. The fundamental prob- 

 lem is to determine the cause for the slow reactions of all or a fraction of 

 an enzyme's SH groups, the total number of such groups being determined 

 by quantitative titrations of the enzyme after complete unfolding. The 

 theories assume either that (I) free SH groups are present in the native cat- 

 alytically active enzyme, but are for some reason unable to react readily 

 with SH reagents, or that (II) the unreactive SH groups are so modified 

 that they are no longer free. 



(I) Free SH groups present in native enzyme 



A. Steric factors impede reaction: the reagent is simply unable to approach 

 the SH group because it is located in a pit or crevice of the enzyme, or ac- 

 tually within the protein structure. 



B. Electrostatic factors impede reaction: the SH group is in the electric 

 field of surrounding groups, this discouraging reactions with reagents of 

 the same charge sign as these groups. 



C. Ionization state impedes reaction: if either the SH or the S~ form reacts 

 preferentially but is not significantly present at the experimental pH, the 

 reaction will be appreciably slowed. 



(II) SH groups are not free in the native enzyme 



A. Present as disulfide groups 



B. Hydrogen bonded to adjacent groups 



C. Present in thiazolidine or thiazoline rings 



D. Reacted with some component of enzyme reaction: for example, acylated, 

 phosphorylated, or complexed with some metal ion. 



The fact that complete opening up or unfolding of the protein structure 

 invariably increases the susceptibility of certain SH groups to attack does 

 not necessarily imply that the groups are in some way within the enzyme, 



