Quantitative determination of SH groups in low- and high-molecular-weight compounds by an electron spin resonance method.

ABSTRACT

To quantitatively determine SH groups in high- and low-molecular-weight compounds, a disulfide biradical (RS-SR), where R is imidazoline residue, has been used. The biradical is shown to participate in a thiol-disulfide exchange reaction with compounds containing SH groups. In this case the ESR spectra of the biradical RS-SR and the resulting monoradical R-SH are different. The reaction of the biradical with cysteine, glutathione, and human serum albumin has been studied using the ESR method and the rate constants kf of this reaction have been calculated. Studies of the pH dependence of kf indicate that the thiol-disulfide exchange occurs by reaction with mercaptidione. Protein human serum albumin and hemoglobin have been modified by RS-SR. It has been shown that the treatment of modified proteins with reduced glutathione leads to removal of the radical from the protein; such modifications are thus reversible. The method proposed has been used to quantitatively determine the SH groups of cysteine and glutathione in mouse and rat blood. The method is shown to coincide within experimental error with the determination of glutathione and cysteine by titration with p-chloromercuribenzoate or reaction with Ellman's reagent. This method allows detection of 10(-6)-10(-7) M SH compounds even in colored and highly absorbing samples. The kinetics of the SH group modification can also be determined, leading to deduction about accessibility of the SH group in protein.