FACTORS •DETERMINING THE REACTIVITIES OF SH GROUPS 643 



(IV) Addition of SH groups to double bonds 



CH— R' R— S— CH— R' 



R— SH +11 ±? I 



CH— R" CH2— R" 



OH 



R— SH + = C— R' ±^ R— S— C— R' 



(This may also be considered as a type of alkylation reaction.) 



Examples: maleate, iV-ethylmaleimide, quinones, acrolein, acetoacetate, 

 and methylglyoxal. 



FACTORS DETERMINING THE REACTIVITIES OF SH GROUPS 



The SH groups of various simple thiols, peptides, and proteins differ mark- 

 edly in reactivity with SH reagents. Although this has been known for many 

 years, the molecular basis for this differential reactivity is poorly understood. 

 In general the reactivity is maximal in simple thiols and minimal in proteins, 

 but in proteins there are usually reactive and unreactive SH groups. Barron 

 (1951) classified SH groups as (a) freely reacting, (b) sluggish, and (c) mask- 

 ed, depending on whether they react readily, slowly, or not at all. Although 

 such a division is often useful in discussing SH groups, there is actually a 

 continuous sequence of groups from highly reactive to unreactive. If a pro- 

 tein is allowed to react with an SH reagent under approximately physiolo- 

 gical conditions, one generally finds that the SH groups disappear at differ- 

 ent rates, perhaps several reacting completely before others are affected. A 

 graded response is clearly seen in the reaction of aldolase with p-chloromer- 

 curibenzoate (Swenson and Boyer, 1957). Ten SH groups react relatively 

 rapidly, a few more slowly, and the rest not at all unless the enzyme is un- 

 folded by high concentrations of urea. Furthermore, the reaction of the first 

 10 SH groups does not alter the enzyme activity, indicating that these 

 groups are not part of, or even too near, the active center, whereas disap- 

 pearance of the more slowly reacting groups abolishes the activity. A similar 

 situation has been observed with urease, which has 5 cysteine residues per 

 molecule, one freely reacting and 4 sluggish, the catalytic activity being 

 affected only by modification of the latter (Hellerman, 1939; Hellerman et 

 al., 1943). Thus porphyrindin, iodoacetamide, and iodosobenzoate react with 

 one SH group but do not inhibit (except at very high concentrations), 

 whereas p-chloromercuribenzoate can combine with another SH group abol- 

 ishing the activity. These examples — and we shall have occasion to discuss 

 many others — illustrate four most important principles: (1) the differential 

 reactivity of enzyme SH groups, (2) the increase in reactivity of many of 

 the SH groups following denaturation, (3) the different reactivities of var- 



