On the Reversible Reaction of Cyanate with Sulfhydryl Groups and the Determination of NH,-terminal Cysteine and Cystine in Proteins*

The present paper is a continuation of studies on the reaction of cyanate with amino acids, peptides, and proteins (1). In the earlier report, emphasis was placed on the speed of the reaction of cyanate with the sulfhydryl groups of cysteine, glutathione, and urea-denatured P-lactoglobulin. The work to be presented in this communication is concerned primarily with a study of the cyanate-sulfhydryl system, and particularly with the equilibrium that has been found to exist between X-carbamylcysteine, cysteine, and cyanate at low temperatures and at pH values near neutrality. The alkali-catalyzed decomposition of S-carbamyl compounds to sulfhydryl compounds and cyanate has been reported previously (cj. Weiss (a), especially Footnote 3). More recently, Ravel et al. (3) have described the reaction of S-carbamylcysteine with alcoholic sodium hydroxide to yield sodium cyanate and cystine. It has now been found, through a consideration of rate constants, that the equilibrium between cyanate, cysteine, and X-carbamylcysteine is such that cyanate can be used as a reversible blocking reagent for sulfhydryl groups. The S-carbamyl group is stable below pH 5 but is removed readily at pH 8. The report of Stark and Smyth (4) that a sulfonic acid is not formed when S ,N-dicarbamylcysteine is oxidized with performic acid has not been confirmed in the present work; S-carbamyland S,N-dicarbamylcysteine do yield sulfonic acids after such oxidation. Consequently, the procedure previously suggested for distinguishing between NHz-terminal cysteine and NHzterminal cystine in a protein causes the eventual formation of cysteic acid in either case. Therefore, a different procedure has now been devised in which the sulfhydryl groups of the protein are alkylated with iodoacetamide before the amino groups are carbamylated; S-carboxymethylcysteine is formed eventually from NH2-terminal cysteine, but not from NHz-terminal cystine.