Rapid capillary electrophoresis analysis of glutathione and glutathione disulfide in roots and shoots of plants exposed to copper.

ABSTRACT

INTRODUCTION: Glutathione and glutathione disulfide can be determined by capillary zone electrophoresis; however, the frequent use of acidic precipitation of protein from samples prior to analysis generates an acidic matrix of strength and pH that may cause changes in the method sensitivity, comigration of species or changes in the equilibria that relate both species in cells or fluids. OBJECTIVE: To optimise electrophoretic conditions for glutathione and glutathione disulfide determination, and to improve pre-analytical treatment for better visualization of the signals of both peptides in an acidic matrix. METHODOLOGY: The method consisted of direct photometric detection at 185 nm and 300 mm borate at pH 7.6 as background electrolyte. The variables under study were voltage applied, injection time, capillary length and electrolyte pH. Seedlings were hydroponically grown and the peptides were extracted with metaphosphoric acid. RESULTS: The resulting acidic matrix was previously treated with the same background electrolyte to prevent comigration and to improve signal resolution. The optimised method showed good reproducibility and linearity, with correlation coefficients above 0.999 and detection limits below 3 microM, and determination of both analytes in less than 3 min. Analyte recovery in the process was in the 88-104% range. The concentration range found in hydroponically grown tomato plants, irrespective of copper level, was 45-100 nmol/g fresh weight for glutathione and below 56 nmol/g fresh weight for glutathione disulfide. CONCLUSION: The results obtained here support the applicability of the method to the fast and simultaneous determination of glutathione and glutathione disulfide in tissue of shoots and roots of plants grown under either normal or stressful conditions.