Reaction of cysteinyl residues with azobenzene-2-sulfenyl bromide

Reaction of cysteinyl residues with azobenzene-2-sulfenyl bromide 

Azobenzene-2-sulfenyl bromide is reported to react selectively with 

The following procedure, applied to egg-white lysozyme, was described by Fontana et al. (1968). 

Protein (40 mg) was dissolved in 2 ml 8 M urea solution, adjusted to pH 8.6 by the addition of 5 % methylamine, and jS-mercaptoethanol (20 pi) added. After 1 hr at room temperature, the protein was precipitated with acetone 1 N HCl (39 1, v/v) at -5°C, separated by centrifugation, and washed with acetone. The protein was dissolved in 2 ml of 25 % (v/v) aqueous acetic acid. Azobenzene-2-sulfenyl bromide (35 mg) was dissolved in 50% aqueous acetic acid (1 ml), and added to the solution of the reduced protein. After 30 min at 22-24°C, the protein derivative was freed of excess reagent by passage through a column of Sephadex G-25 (90 x 1 cm), equilibrated and developed with 0.2 M acetic acid, and lyophilized. 

The modified protein was dissolved in 0.1 N HCl (2 mg/ml) and an aliquot (0.3 ml) added to 2.7 ml of 8 M urea adjusted with HCl to pH 1. The concentration of azobenzene-2-sulfenyl residues (e= 16700 at 353 nm) was determined spectrophotometrically, and extent of modification calculated by comparing this concentration with the concentration of protein in the 0.1 N HCl solution, determined by amino acid analysis on an appropriate aliquot. 

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