37] Reaction of protein sulfhydryl groups with Ellman's reagent

Abstract

Publisher Summary This chapter discusses a reaction of protein sulfhydryl groups with Ellman's reagent. The reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is developed by Ellman as a sulfhydryl reagent. DTNB is found to be a sensitive tool for the assay of thiol groups in tissues, body fluids, and proteins. Ellman described a method for its synthesis. DTNB is an aromatic disulfide, and, since it has a higher standard oxidation-reduction potential than aliphatic analogs, it will react with aliphatic thiols by an exchange reaction to form a mixed disulfide of the protein and 1 mole of 2-nitro-5-thiobenzoate per mole of protein sulfhydryl group. Sulfhydryl-disulfide exchange reactions between disulfide compounds with sulfur directly attached to aromatic groups and simple alkyl mercaptans should go to completion. Protein sulfhydryl (SH) groups may behave similarly to simple alkyl mercaptans unless steric factors interfere with the course of reaction. The sulfhydryl groups in proteins exhibit variable reactivity toward DTNB owing to steric factors. Determination of total sulfhydryl content requires that the protein be denatured, preferably with sodium dodecyl sulfate.