640 4. SULFHYDRYL REAGENTS 



have ESH, ESSG, ESSE, GSSG, and GSH (where E represents the enzyme) 

 present, perhaps only ESH being catalytically active and the forms ESSG 

 and ESSE protected from SH reagents. Although it is usually thought that 

 only the SH form can react with most SH reagents, it is possible that di- 

 sulfides are occasionally reactive; aryl arsinites, for example, can exert a 

 nucleophilic displacement on the disulfide bond: 



OH 

 RSSR + R'AsO(OH)- ±^ RSAsR' + RS~ 



O 



However, such reactions are probably slower than with free SH groups. 

 The new reagent, dithiothreitol (HS— CH2— CHOH— CHOH— CHg— SH), 

 which has a low redox potential ( — 0.33 v at pH 7), is highly water-soluble 

 and reduces protein disulfide groups (Cleland, 1964). It was suggested that 

 it might be valuable in protecting enzyme SH groups, having several ad- 

 vantages over the ones commonly used, and could also be applied for the 

 purpose of maintaining enzymes in the SH state for inhibition studies. 



Hydrogen bonding by sulfur should be mentioned since it must play a 

 role in both intra- and intermolecular interactions of the SH group, but 

 most of the data we have derives from studies of the small thiols and there 

 is very little information on hydrogen bonding of protein SH groups. Boyer 

 (1959) has presented the evidence for the occurrence of hydrogen bonds to 

 sulfur in a variety of compounds. Sulfur does not form hydrogen bonds as 

 readily as oxygen or nitrogen, since it is less electronegative (as indicated 

 by the smaller dipole moment of the S — H bond compared to the — H and 

 N — H bonds) (Table 1-6-1). However, there is evidence for intramolecular 

 S — H • • • and S — H • • • N bonds in cysteine and its peptides, and Benesch 

 et al. (1954) have advanced hydrogen bonding to explain some of the dif- 

 ferent reactivities of simple thiols. It is possible that hydrogen bonding of 

 enzyme SH groups can modify their susceptibilities to various SH reagents. 



Detection and Determination of Enzyme SH Groups 



Valuable reviews of the general methods for the determination of SH 

 groups in proteins and enzymes have been provided by HeUerman and 

 Chinard (1955) and R. Benesch and R. E. Benesch (1962). Some of the 

 most reliable methods involve the use of mercurials (to be discussed later, 

 pages 762 and 766). Here we shall mention only a few of the more recently 

 developed reagents which may be applicable in inhibition studies. 



Bis(2?-nitrophenyl)disulfide reacts with thiols at pH 8 to form 1 mole of 

 p-nitrophenol per mole of thiol, and this anion, being highly colored, can 

 be used to determine the thiol concentration (Ellman, 1959). However, this 



