Characterization of a new sulfhydryl group reagent: 6,6′-Diselenobis-(3-Nitrobenzoic acid), a selenium analog of Ellman's reagent

A new reagent, 6,6′-diselenobis-(3-nitrobenzoic acid) (DSNB) has been synthesized and is shown to be useful for quantitative estimation of sulfhydryl groups in proteins. This reagent, which is a selenium analog of Ellman's reagent, reacts specifically and quantitatively with thiol groups of proteins to yield a selenenyl sulfide and the dianion of 3-nitro-6-selenobenzoic acid. The molar absorption coefficient of the chromophoric dianion is 10,000 at 432 nm in dilute aqueous solutions. The titration can best be performed at pH 8.20 where >98% of 3-nitro-6-selenobenzoic acid is in the form of the intensely colored dianion. Sulfhydryl content determinations by this reagent of reduced and denatured ribonuclease, reduced and denatured lysozyme, native papain, and native and denatured thymidylate synthetase are compared with those from corresponding 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) titrations. The reagent was found to inactivate thymidylate synthetase, an enzyme with essential sulfhydryl groups. Unlike DTNB which undergoes alkaline decomposition of pH values greater than 9, DSNB was found to be stable to hydrolysis, even in 0.05 m NaOH.