Sulfenyl halides react selectively in acidic solvents with cysteine and tryptophan residues in polypeptides and proteins. Tryptophan is converted into a derivative with a thioether function a t the 2 position of the indole nucleus. Cysteine is converted into a n unsymmetrical disulfide. The halides which were studied included 2-nitro-(NPS-CI), 4-nitro-(pNPS-CI), and 2,4-dinitrophenylsulfenyl chloride (DNPS-CI). By reduction of the mixed disulfides with P-mercaptoethanol, thioglycolic acid, or sodium borohydride, the original thiol group is easily restored. The unsymmetrical disulfides are stable under acidic conditions. In alkaline solution, I n the preceding paper it was shown that sulfenyl halides are selective and mild reagents for tryptophan and cysteine (Scoffone et al., 1968). The present paper describes the results of the reaction of sulfenyl halides with cysteine in model compounds and in proteins.Sulfenyl halides react very rapidly and stoichiometrically with the thiol group of cysteine, leading to an unsymmetrical disulfide. The reaction, which occurs readily in acidic solvents (aqueous acetic acid), was applied t o cysteine derivatives and to reduced glutathione, and the corresponding disulfides were isolated in good yield. This modification of cysteinyl residues is easily reversed by employing the reducing reagents (e.g., P-mercaptoethanol, thioglycolic acid, and sodium borohydride) extensively used in protein chemistry for the reduction of disulfide bonds.The suitability of sulfenyl halides in a reversible modification of SH groups was tested by treating the reduced ribonuclease (Anfinsen and Haber, 1961) with 2-nitro-(NPS-CI)' and 4-nitrophenylsulfenyl chloride (pNPS-CI). The NPS protein or pNPS protein was then further reduced with P-mercaptoethanol in 8 M urea. After purification on Sephadex G-25, air oxidation Padova. l Abbreviations used: NPS, 2-nitrophenylsulfe~iyl; pNPS, 4-nitrophenylsulfenyl; DNPS, 2,4-dinitrophenylsulfenyl; DMF, dimethylformamide; NPS-RNase and pNPS-RNase, derivatives of reduced ribonuclease treated with 2-nitro-or 4-nitrophenylsulfenyl chloride. The amino acids, peptides, and peptides derivatives are of L configuration. The abbreviations are those recommended by the IUPAC-IUB Commission on Biochemical Nomenclature, Biochemistrj. 5, 1445 (1966). 980 however, they are decomposed, releasing the nitrothiophenol moiety quantitatively. The latter can be assayed by spectrophotometry, thus affording a direct measure of the free cysteine in a protein. The mildness of this reversible reaction with cysteine residues was checked with reduced ribonuclease (RNase). After reaction of the reduced enzyme with 2-nitro-or 4-nitrophenylsulfeny1 chloride in 50 acetic acid, the NPS-RNase and pNPSRNase were isolated and subjected to further reduction with P-mercaptoethanol in 8 M urea. After purification on Sephadex G-25 and air oxidation, the recovered enzyme retained 70-80% of its activity toward RNA.produces the enzyme without significant loss of activity, proving the mildness of the reaction ...