REACTIONS WITH PROTEINS 767 



ever, Flesch and Kun (1950) found that the addition of strong acid intensifies 

 the color markedly. It has been claimed that this mercurial is as specific as 

 PM for SH groups, but one wonders if this complex molecule does not 



N=N^ y-Hg 



/)-Mercuriphenylazo-/:i-naphthol 



through other groups occasionally react with various tissue components 

 (/5-naphthol derivatives being fairly potent enzyme inhibitors), although 

 previous treatment of the tissue with Hg++ or iodoacetamide is said to 

 prevent staining. Aqueous solutions of thiols, proteins, or tissue homogenates 

 are shaken with an amyl acetate solution of the mercurial dye, and a red 

 precipitate slowly forms in the aqueous phase as the reaction proceeds; the 

 amount of precipitate is proportional to the number of SH groups and can 

 be determined colorimetrically after centrifuging and redissolving in acid 

 solution. Fragments of dehydrated tissues may also be placed in butanol 

 or propanol solutions of the mercurial for several hours, and the staining 

 demonstrated histologically (Bennett, 1951). Bennett ran controls with phe- 

 nylazo-/5-naphthol to determine if this portion of the molecule contributed 

 to the binding, and generally found little or no staining. This mercurial 

 has been used to investigate thiol distribution in muscle (Bennett, 1948 b), 

 skin (Mescon and Flesch, 1952), and a variety of other tissues (Bennett, 

 1951). 



Another colored mercurial, 4-mercuri-4'-dimethylaminoazobenzene, has 

 been used by Horowitz and Klotz (1956) to determine protein SH groups. 

 The solubility in water is so low that colorimetric determinations cannot 



4-Mercuri-4'-dimethylaminoazobenzene 



be made, but it dissoves sufficiently in 100 raM glycine (due to the forma- 

 tion of a glycinate complex) that reactions with SH groups in aqueous 

 medium can be carried out. However, it is also possible to determine the 

 amount of the mercurial removed from heptanol when shaken with an 

 aqueous solution containing the protein, although equilibrium usually re- 

 quires several hours. The specificity of reaction appears to be satisfactory, 



