CHEMICAL PROPERTIES OF SH GROUPS 641 



reagent is poorly soluble in water, so carboxyl groups were introduced to 

 solubilize it; this compound is 5,5'-ditliiobis(2-nitrobenzoate) and reacts 



0,N {' V — S— S — V ) — NO; 



5, 5'-Dithiobis (2-nitrobenzoate) 



with thiols and SH groups in the blood and tissues. Another water-soluble 

 reagent for free SH groups is 2,2'-dicarboxy-4,4'-diiodoaminoazobenzene, 

 which was shown to react only with the SH groups on denatured meromyosin 

 (Fasold et al., 1964). The number of SH groups reacted can be determined 



coo' 



I— CHi— CONH-Y^ \^N=N^/ ^V-NHCO— CH2— I 



"ooc 



2, 2'-Dicarboxy-4, 4'-diiodoaminoazobenzene 



spectrophotometrically because of the chromogenic azo link. A yellow SH 

 reagent, A^-(4-dimethylamino-3,5-dinitrophenyl)maleimide, was introduced 

 by Witter and Tuppy (1960) and found to react with the free SH groups 

 of seralbumin. The treated protein could be hydrolyzed with pepsin and 

 the A'-(4-dimethylamino-3,5-dinitrophenyl)succinimido-cysteine peptides 

 isolated by means of their yeUow color. This reagent was used by Gold and 

 Segal (1964) to obtain information on the nature of the active site of 3- 

 phosphoglyceraldehyde dehydrogenase. Following pepsin treatment the sin- 

 gle hexapeptide - Ala-Ser-(DDPS-Cys)-Thr-Thr-AspNH2 - w^as found to 

 contain essentially aU the color. This provides evidence that the three active 

 sites on the enzyme are similar in structure, at least in part, and that the 

 reactive SH groups are not those of glutathione, which occurs on the enzyme. 



O.N 



0,N 



A/-(4-Dimethylamino-3, 5- 

 dinitrophenyl) maleimide 



Such reagents would not be particularly useful for the inhibition of SH en- 

 zymes because of their bulky structure and the presence of a variety of 



