Formation of δ1-acetoxytryptophan-62 in the oxidation of tryptophan-62 of hen egg-white lysozyme by N-bromosuccinimide in acetate buffer

Abstract

The reaction of equimolar amounts of N-bromosuccinimide and hen egg-white lysozyme in acetate buffer, under the conditions of Hayashi et al. yields a protein mixture that has a time-dependent 13C-NMR spectrum. The initial natural-abundance 13C-NMR spectrum indicates the presence of about equal amounts of [oxindolealanine-62]lysozyme and [.delta.1-acetoxytryptophan-62]lysozyme. The latter converts to [oxindolealanine-62]lysozyme with a half-life of about 2 days at 25.degree. C and pH 3.9. Two observations indicate that the source of the acetyl group of .delta.1-acetoxytryptophan-62 is the acetate buffer. First, the spectrum of a lysozyme sample treated with N-bromosuccinimide in the presence of [1-13C]acetate yields a very strong acetyl ester carbonyl resonance. The time dependence of the intensity of this resonance yields a half-life of 44 h for [.delta.1-acetoxytryptophan-62]lysozyme. Second, the initial natural-abundance 13C-NMR spectrum of a lysozyme sample treated with N-bromosuccinimide in the absence of acetate indicates essentially complete conversion of tryptophan-62 into oxindolealanine.