S-nitrosation of serum albumin: Spectrophotometric determination of its nitrosation by simple S-nitrosothiols

The transfer of nitroso groups from S-nitroso-L-cysteine (1) and six other simple S-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 6400 M(-1) s(-1) obtained for the reaction of (2) with S-nitrosomercaptoethylamine is similar to 10 times faster than that for its reaction with (1), similar to 40 times faster than that for its reaction with S-nitrosoglutathione, and consistent with Cys 34 being located in a narrow crevice in close proximity to an anionic charge. (C) 1996 Academic Press, Inc.