Manual and flow-injection spectrophotometric assay of thiols, based on their S-nitrosation

A general assay procedure for a wide variety of thiols is described. The technique has three steps: (1) formation of S-nitrosothiols with nitrous acid, (2) destruction of the excess of nitrous acid, (3) hydrolysis of the S-nitrosothiols with mercuric ions and subsequent formation of an azo-dye by means of the nitrous acid liberated. Both manual and flow-injection analysis (FIA) versions of the assay are described. Since the final step of the assay does not depend on the thiol assayed but only on the reaction of nitrous acid to form azo-dyes, the calibration graphs should be identical for all thiols. The manual system is about four times as sensitive as the FIA system, but the latter permits a high sample throughput and shows significantly lower sensitivity to interference by tryptophan. Though this general technique cannot be used for the assay of many sterically hindered thiols, it can be used for the assay of some protein thiol groups.