Estimation of Disulfide Bonds Using Ellman’s Reagent

Abstract

Ellman’s reagent 5,5′ -dithiobis(2-nitrobenzoic acid) (DTNB) was first introduced in 1959 for the estimation of free thiol groups (1). The procedure is based on the reaction of the thiol with DTNB to give the mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB) which is quantified by the absorbance of the anion (TNB^2-) at 412 nm. The reagent has been widely used for the quantitation of thiols in peptides and proteins. It has also been used to assay disulfides present after blocking any free thiols (e.g., by carboxymethylation) and reducing the disulfides prior to reaction with the reagent (2, 3). It is also commonly used to check the efficiency of conjugation (4) of sulf-hydryl-containing peptides to carrier proteins (such as maleimide activated keyhole limpet haemocyanin, KLH) in the production of antibodies and to ensure that cysteine labeled sythetic peptides are coupled to affinity resins.