A modification of the Ellman procedure for the estimation of protein sulfhydryl groups

The reaction of the Ellman reagent [5,5′-dithiobis-(2-nitrobenzoic acid)] with a protein sulfhydryl group yields 1 mole of thiophenylated protein and 1 mole of thiophenylate anion, when the reaction is carried out above pH 7.0. The quantity of thiophenylate anion liberated in this reaction is then determined by light absorption measurement at 412 mμ. In the present paper, this technique is modified so that the thiophenylated protein is isolated from the reaction mixture. The amount of thionitrobenzoic acid bound to the protein is then determined. Using this principle, sulfhydryl analyses have been successfully made on (1) a sulfhydryl-containing protein in the presence of a reduced thiol; (2) heme proteins; (3) an insoluble protein; and (4) a protein rendered insoluble by denaturation. The classical Ellman procedure cannot be used for assays of free sulfhydryl groups on any of these proteins.