Sulfenyl Halides as Modifying Reagents for Polypeptides and Proteins

The determination of tryptophan and cysteine content in proteins by means of the selective reaction of sulfenyl halides with these amino acids has been carefully investigated. By reaction with 2‐ or 4‐nitrophenylsulfenyl chloride in aqueous acetic or formic acid, the nitrophenyl chromophore is covalently bound to the tryptophan and the cysteine residues of the protein, giving a thioether and a mixed disulfide respectively. The cysteine content has been determined spectrophotometrically at 450 nm from the 4‐nitrothiophenol released from the mixed disulfides of the cysteine residues after exposure of the 4‐nitrophenylsulfenyl chloride‐labelled protein in 0.1 N NaOH (at this wavelength the corresponding chromophore covalently bound to tryptophan does not absorb). Tryptophan has been estimated in the sulfenylated proteins by spectrophotometric determinations at 365 or 328 nm of the 2‐ and 4‐nitrophenylsulfenyl chromophores (ɛ M 4000 and 11700 respectively), following removal of the yellow label from the cysteine residues, by dissolving the modified protein in 0.1 N NaOH. The procedure has been shown to be accurate for both amino acids with a wide variety of proteins.