QUANTITATIVE-DETERMINATION AND REVERSIBLE MODIFICATION OF THIOLS USING IMIDAZOLIDINE BIRADICAL DISULFIDE LABEL

Earlier we reported an ESR method of quantitative determination of sul fhydryl groups. The method is based on the application of the imidazol ine biradical disulfide label, R1S-SR1, which participates in the reac tion of thiol-disulfide exchange followed by dramatic changes in ESR s pectra. One of the disadvantages of the application of R1S-SR1 at phys iological conditions is the requirement of excess of the biradical com pared with thiol content which results in the consumption of the thiol s and irreversible damage of the system under study. In the present pa per 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 bir adical disulfide, R1S-SR1, such as high sensitivity down to 1 mu M of thiols even in opaque samples and could possibly be used for reversibl e modification of proteins and enzymes. The particular properties of t he 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 p hysiological conditions. The latter makes it possible to use ESR spect roscopy for non-invasive thiol measurements in biological systems, in vivo applications included. (C) 1997 Elsevier Science B.V.