Quantitative determination and reversible modification of thiols using imidazolidine biradical disulfide label.

Earlier we reported an ESR method of quantitative determination of sulfhydryl groups. The method is based on the application of the imidazoline biradical disulfide label, R1S-SR1, which participates in the reaction of thiol-disulfide exchange followed by dramatic changes in ESR spectra. One of the disadvantages of the application of R1S-SR1 at physiological conditions is the requirement of excess of the biradical compared with thiol content which results in the consumption of the thiols and irreversible damage of the system under study. In the present paper we propose imidazolidine biradical disulfide reagent, R2S-SR2, for ESR determination of thiols and provide an experimental basis for its application. This label has the advantages of the previously used biradical disulfide, R1S-SR1, such as high sensitivity down to 1 microM of thiols even in opaque samples and could possibly be used for reversible modification of proteins and enzymes. The particular properties of the R2S-SR2 are pH-sensitivity of its ESR spectrum, higher stability of the imidazolidine radical fragment towards biological reductants and low concentration of the label sufficient for thiol determination at physiological conditions. The latter makes it possible to use ESR spectroscopy for non-invasive thiol measurements in biological systems, in vivo applications included.