Capillary zone electrophoretic detection of biological thiols and their S-nitrosated derivatives

Reduced thiols (RSH) react with certain oxides of nitrogen to yield S-nitroso thiols (RSNO) possessing smooth muscle relaxant and platelet inhibitory properties. Nitrosated derivatives of the biological thiols--glutathione, cysteine, and homocysteine--have therefore been considered as bioactive intermediates in the metabolism of organic nitrates and the endothelium-derived relaxing factor with properties of nitric oxide. As yet, however, there is no established chemical method for identifying the biological S-nitroso thiols and, consequently, little is known of their distinguishing chemical characteristics or biochemistry. In this study, we demonstrate for the first time a simple, rapid, and reproducible method for separating these thiols from their S-nitrosated derivatives using capillary zone electrophoresis. Cysteine, homocysteine, and glutathione were separated from one another and from their corresponding disulfides in 0.01 M phosphate buffer, pH 2.5, by capillary zone electrophoresis and absorbance detection at 200 nm with measured elution times of 5.92-16.15 min; corresponding S-nitroso thiols were selectively detected at 320 nm and eluted at 2.50-18.20 min. These data support the specificity and reproducibility of this technique for separation and identification of thiols, disulfides, and S-nitroso thiol derivatives.