Reaction of N-bromosuccinimide with lysozyme.

Abstract

1.1. Amino acid analysis of lysozyme treated with N-bromosuccinimide at pH 4 and 5.5 revealed extensive destruction of tyrosine and histidine at reagent concentrations at which the spectral technique indicated incomplete reaction with tryptophan. Rotatory dispersion and viscosity measurements demonstrated the occurrence of a conformational change in lysozyme after reaction at these N-bromosuccinimide levels.2.2. Amino acid analysis for tryptophan after alkaline hydrolysis of N-bromosuccinimide-treated lysozyme showed higher degrees of oxidation of this amino acid than revealed by the spectral procedure. In contrast with the observations made spectrally, no pH dependence of oxidation was evident.3.3. The apparent pH dependence of N-bromosuccinimide reactivity, as well as the apparent differential reactivity of the six tryptophans, is most likely an artifact of the spectral procedure arising from oxidation of tyrosyl groups.4.4. The lack of specificity of the reaction of N-bromosuccinimide with respect to tryptophan groups of lysozyme, the induction of a conformational change on reaction and the lack of correspondence of spectral and amino acid analysis data suggests that caution must be exercised in the use of N-bromosuccinimide as a reagent for determining the extent of ‘exposure’ of side chains of native proteins. 1. Amino acid analysis of lysozyme treated with N-bromosuccinimide at pH 4 and 5.5 revealed extensive destruction of tyrosine and histidine at reagent concentrations at which the spectral technique indicated incomplete reaction with tryptophan. Rotatory dispersion and viscosity measurements demonstrated the occurrence of a conformational change in lysozyme after reaction at these N-bromosuccinimide levels. 2. Amino acid analysis for tryptophan after alkaline hydrolysis of N-bromosuccinimide-treated lysozyme showed higher degrees of oxidation of this amino acid than revealed by the spectral procedure. In contrast with the observations made spectrally, no pH dependence of oxidation was evident. 3. The apparent pH dependence of N-bromosuccinimide reactivity, as well as the apparent differential reactivity of the six tryptophans, is most likely an artifact of the spectral procedure arising from oxidation of tyrosyl groups. 4. The lack of specificity of the reaction of N-bromosuccinimide with respect to tryptophan groups of lysozyme, the induction of a conformational change on reaction and the lack of correspondence of spectral and amino acid analysis data suggests that caution must be exercised in the use of N-bromosuccinimide as a reagent for determining the extent of ‘exposure’ of side chains of native proteins.