DISULFIDES 661 



tributed to SH oxidation, but because of the molecular complexity of most 

 dyes it would be very unlikely that specific effects on SH groups could be 

 obtained. Inhibitions by dyes will be discussed in a separate chapter. 



DISULFIDES 



Cystine oxidizes certain protein SH groups and was first used for the de- 

 termination of these groups by Mirsky and Anson (1935). They found that 

 those protein SH groups reacting with nitroprusside are completely oxidized 

 by cystine. It was stated by Mirsky and Anson, and has been restated by 

 others, that cystine is one of the most specific oxidants of protein SH groups. 

 One disadvantage of cystine is its low solubility; dithioglycolate was sug- 

 gested as superior in this regard but has been used very little. Another dis- 

 advantage is perhaps that the oxidation-reduction potential of the cystine- 

 cysteine couple is not high enough to oxidize all the SH groups, which, if it 

 is assumed that the mean potential of protein SH groups is similar to free 

 cysteine, must be true. A further complication is the formation of mixed 

 disulfides (see page 639): 



E— SH + R— S— S— R ±5 E— S— S— R + R— SH 



which is not the simple oxidation of enzyme SH groups previously suppos- 

 ed, a portion of the disulfide being bound to the enzyme. 



A few results on enzyme inhibition are summarized in Table 5-1. The in- 

 hibitions are not to be taken quantitatively because the conditions and the 

 incubation times vary greatly. Particularly important is the duration of 

 contact between the enzyme and the disulfide, since in almost all instances 

 the reaction has been found to proceed very slowly. Rapkine (1938) found 

 that phosphoglyceraldehyde dehydrogenase requires up to 5 hr for maximal 

 inhibition by GS,SG, and Hopkins et al'. (1938) showed that the inhibition 

 of succinate dehydrogenase develops steadily over 2 hr, and probably con- 

 tinues after that time. Whether this is due to the slowness of the oxidation 

 of enzyme SH groups or to secondary factors is not known. One reason for 

 the lack of inhibition of certain enzymes by disulfides may well be that suf- 

 ficient time was not allowed for reaction. Such slow reactions limit the use 

 of the disulfides for either SH group determination or enzyme studies. In- 

 sufficient examination of the reversibility of disulfide inhibitions by reduc- 

 tion also makes it difficult to evaluate the mechanism. Rapkine (1938) found 

 that both GSH and cysteine reactivate GSSG-inhibited phosphoglyceral- 

 dehyde dehydrogenase, and Hopkins and Morgan (1938) obtained similar 

 results with succinate dehydrogenase, but reversibility has not been at- 

 tempted in most work. Many enzymes require SH groups for activity but 

 others are active only when disulfide bonds are formed. Cytochrome oxidase 



