Effects of Oxidation of Tryptophan Residues in Thioredoxin from Escherichia coli by N-Bromosuccinimide

Both tryptophan residues of Escherichia coli thioredoxin were chemically modified to oxindole by a 6-fold molar excess of N-bromosuccinimide at pH 4.0. The thioredoxin derivative with oxindole side chains at residues 28 and 31 was enzymatically inactive with thioredoxin reductase and ribonucleotide reductase. It showed complete lack of tryptophan fluorescence, but gave full cross-reaction with antithioredoxin antiserum in double immunodiffusion and precipitin tests. Tryptophan fluorescence titration experiments of the reaction of thioredoxin with N-bromosuccinimide strongly suggested that 1 tryptophan residue could be selectively modified. Addition of a 3-fold molar excess of reagent at pH 4.0 modified a residue which had a quenched fluorescence emission in both the oxidized and reduced form of thioredoxin. This residue was not essential for activity of thioredoxin with thioredoxin reductase. The 2nd tryptophan residue had a quenched tryptophan fluorescence in the oxidized form of thioredoxin but dominated the emission spectrum of the reduced form.