Modification of the Single Tryptophan Residue of Staphylococcal Nuclease by a New Mild Oxidizing Agent

Abstract

Abstract The single tryptophan residue of staphylococcal nuclease was selectively modified with the use of a new bromine-positive releasing compound obtained by reaction of 2-(2-nitrophenylsulfenyl)-3-methylindole with N-bromosuccinimide. The oxidizing properties of the reagent for amino acid side chains of proteins are much more selective than those of N-bromosuccinimide. Cleavage of the tryptophanyl peptide bond also can be obtained (yields up to 59% on model compounds) with use of relatively high amounts of reagent and longer reaction times. The nuclease derivative, with an oxindole side chain at Residue 140, showed complete lack of tryptophan fluorescence, and the molar absorptivity at 280 nm was decreased in proportion to the loss of tryptophan. Ultraviolet circular dichroism was consistent with loss of helical structure. However, the modified enzyme retained 18% DNase and 12% RNase activity and cross-reacted with native nuclease in agar immunodiffusion against antinuclease antiserum. When the protein was incubated with a large excess of reagent for 28 hours at room temperature, selective cleavage of the tryptophanyl peptide bond was observed, and the COOH-terminal nonapeptide of nuclease was isolated in 15% yield.