S-Nitrosation of Serum Albumin: Spectrophotometric Determination of Its Nitrosation by SimpleS-Nitrosothiols

Abstract

The transfer of nitroso groups fromS-nitroso-L-cysteine (1) and six other simpleS-nitrosothiols to Cys 34 of bovine serum albumin (2) has been followed using Ellman's reagent, 5,5′-dithio-bis (2-nitrobenzoate) (3), to detect the resulting thiols. The described method utilizes the low reactivity of (3) with (2) and the high extinction coefficient of 2-nitro-5-thiobenzoate produced upon its reaction with thiols to follow the transfer of nitroso moieties at low concentrations where other procedures are not feasible. A second-order rate constant of 6400M−1s−1obtained for the reaction of (2) withS-nitrosomercaptoethylamine is ∼10 times faster than that for its reaction with (1), ∼40 times faster than that for its reaction withS-nitrosoglutathione, and consistent with Cys 34 being located in a narrow crevice in close proximity to an anionic charge.