Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent.

Studies of protein-bound (PB-SH) and nonprotein bound sulfhydryl group (NP-SH) concentrations in tissues under various conditions is a prerequisite to understanding the role of sulfhydryls in living organisms. Many methods have been developed for the measurement of sulfhydryl groups, but these have been concerned chiefly only with the estimation of NP-SH in biological fluids or total SH groups (T-SH) in purified protein or other substances. The reviews on SH determinations by Chinard and Hellerman (1) and by Benesh and Benesh (2) give excellent and comprehensive discussions of the general problems involved in sulfhydryl determinations. However, few investigators (3-5) have discussed the concomitant estimation of PB-SH, NP-SH, and T-SH in biological materials and none of the methods used is convenient for the routine estimation of sulfhydryls in these fractions. In addition, Jocelyn’s method (4) has been unsatisfactory in our hands because of poor reproducibility and recoveries. The purpose of this communication is to report a simple spectrophotometric method for the routine concomitant determination of sulfhydryl groups in PB-SH, NP-SH, and T-SH fractions in various tissues. These spectrophotometric procedures are based on the method of Ellman (6, 7), who reported that 5,5’-dithiobis(2,-nitrobenzoic acid) is reduced by SH groups to form 1 mole of 2-nitro-5-mercaptobenzoic acid per mole of SH. The reaction is: