Reversible Chemical Modification of the Tryptophan Residues of Thioredoxin from Escherichia coli B

Treatment of thioredoxin from Escherichia coli with formic acid saturated with HCl by the method of Previero et al. (1967), resulted in a redshift of the ultraviolet spectrum of the protein consistent with chemical modification of both tryptophans to 1‐formyl‐tryptophanyl residues. Concomitantly with the formylation the protein lost its redox activity in the enzymatic reactions with thioredoxin reductase and ribonucleotide reductase. Incubation of thioredoxin in formic acid alone had no effect on the spectrum or biological properties of the protein. Gel chromatographic experiments on Sephadex G‐50 and G‐75 showed that the formylated thioredoxin was eluted well apart from native thioredoxin and existed in the form of high molecular weight aggregates. This indicated that major conformational changes of thioredoxin accompanied the conversion of the hydrophobic tryptophan residues into the more hydrophilic 1‐formyl‐tryptophans. Treatment of 1‐formyl‐tryptophanyl thioredoxin at pH 9.5 in 6 M guanidine hydrochloride for 16 h resulted in almost complete regeneration of the ultraviolet spectrum of native thioredoxin. Also regenerated was more than 50% of thioredoxin activity in the reaction with thioredoxin reluctase. The results of this reversible chemical modification strongly indicate that the two tryptophan residues play a major role in maintaining the biologically active conformation of thioredoxin from E. coli.