FACTORS DETERMINING THE REACTIVITIES OF SH GROUPS 645 



since denaturation can also dissolve disulfide bonds, hydrogen bonds, ring 

 structures, and other possible chemical interactions of the SH groups. As 

 long as one studies only a single SH reagent, it is easy to postulate a reason- 

 able mechanism for the unreactivity of particular SH groups. For example, 

 if one finds that iodoacetate does not alkylate an enzyme SH group, the 

 group may be thought of as sequestered within the protein structure, but 

 if subsequent work shows that p-chloromercuribenzoate reacts readily with 

 this group, this hypothesis must be abandoned inasmuch as j^-chloromer- 

 curibenzoate is a larger molecule than iodoacetate. Likewise, postulating 

 that negative charges surround the SH group, preventing the approach of 

 iodoacetate, will not be valid if p-chloromercuribenzoate is effective, since 

 both of these reagents are negatively charged, as has been pointed out by 

 Boyer (1959) in perhaps the best discussion of differential SH group reac- 

 tivity. It is certainly likely that steric and electrostatic factors are occa- 

 sionally important, but one must demonstrate some correlation of the un- 

 reactivity of the SH groups with the properties of a variety of SH reagents. 

 Haurowitz and Tekman (1947) believed that protein SH groups are often 

 inaccessible to reagents because of the tightly folded nature of the polypep- 

 tide chains, rather than chemically combined, because unfolding is accom- 

 panied by the appearance of reactivity in other than SH groups, e.g. phe- 

 nolic groups. Although this is suggestive, it is not proof for the inaccessibi- 

 lity theory. Unreactivity due to an unfavorable ionization state has per- 

 haps been insufficiently considered, particularly for inhibitors such as iodo- 

 acetate, and there is no question but that the very low concentration of S~ 

 near neutrality for some SH groups must be important. 



Turning to the second group of theories, no one denies that disulfide 

 groups occur in some enzymes, but that this generally cannot explain the 

 differential reactivity of SH groups is obvious. Indeed, one finds a wide 

 range of reactivities in simple thiols where cryptic exclusion or disulfide 

 bonding may be eliminated. Benesch et al. (1954) not only demonstrated 

 different nitroprusside reaction rates with various biologically important 

 thiols, but showed that urea increases the reactivity of the more slowly 

 reacting SH groups, just as it does in proteins. This was interpreted in 

 terms of the breaking of hydrogen bonds and thus the initial sluggishness 

 of reaction as due to hydrogen bonding of the SH groups to adjacent amino 

 or peptide groups. This may occur in a cysteine peptide in the following 

 way: 



H,C "H H,C H 



^11 "I i 



R,— CONH-HC— CO— N— R, R— CONH— HC— CO— NH— R, 



the hydrogen donator depending on the pH. We have seen that SH groups 

 form only weak hydrogen bonds (page 640), so that it is likely that this 



