[Quantitative determination and reversible modification of sulfhydryl groups in low and high molecular weight compounds using a biradical spin marker]

A new method for quantitation of sulfhydryl groups of low and high molecular weight compounds is proposed. The method is based on the use of a biradical spin label carrying a disulfide bond, RS-SR, where R is the imidazoline radical. It was found that this biradical is involved in the reaction of thiol-disulfide exchange with thiols; the EPR spectra of the original biradical and monoradical products differ essentially. This circumstance made it possible to determine the bimolecular rate constant for the biradical interaction with cysteamine, cysteine, glutathione and human serum albumin. The method was used for measuring glutathione and cysteine levels in murine and rat blood and for assaying the insect acetylcholine esterase activity and reversible inhibition of NADPH-cytochrome P-450 reductase. The method is marked for a high sensitivity (10(-6)-10(-7) M) towards sulfhydryl groups and allows the determination of thiol groups in coloured and nontransparent solutions.