Simultaneous Determination of Glutathione and Glutathione Disulfide in an Acid Extract of Plant Shoot and Root by Capillary Electrophoresis

Abstract

This study describes the fast and simultaneous determination of glutathione and glutathione disulfide by Capillary Zone Electrophoresis in plant extracts of shoot and root of tomato plants. Frequent use of acidic precipitation of protein 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. In this study, the resulting acidic matrix was previously treated with the same background electrolyte to prevent comigration and to improve signal resolution. Optimization of some parameters of the technique allowed the determination of both analytes in less than three minutes. The optimized method showed good reproducibility and linearity, with correlation coefficients above 0.999 and detection limits below 3 µM for both peptides. Analyte recovery in the process was in the 88-104% range. The concentration found in tomato plants hydroponically grown in the absence of stress factors was in the 51-100 nmol g -1 range, fresh weight for GSH and 5-32 nmol g -1 range, fresh weight for GSSG.

Phenotyping methods to assess heat stress resilience in grapevine

“…Acid extraction can also be performed with m-phosphoric acid, as seen for AA. Other methods applied for the determination of GSH in plant tissues are derivatization procedures coupled with HPLC methods with UV or fluorometric detectors (Siller-Cepeda et al, 1991;Chen et al, 1999), HPLC methods coupled with MS (Guček et al, 2002;Rellán-Álvarez et al, 2006), or CE techniques (Davey et al, 1997;Herrero-Martínez et al, 2000;Mendoza et al, 2008). Although focused on the medical field, a general and useful review on HPLC and electrophoresis methods for GSH and GSSG is presented by Camera and Picardo (2002).…”

Recent advances in CE and CEC of peptides (2007–2009)

“…The best separation was achieved within less than 30 min using a 10 mM phosphate BGE, pH 2.8, in bare FS capillary (75 μm id, 47/40 cm) at 18°C and 15 kV separation voltages. CZE with UV‐detection at ultralow UV detection wavelength 185 nm enhancing sensitivity of this detection mode has been developed for fast simultaneous analysis of GSH and GSSG in the roots and shoots of tomato plants grown under normal or stressful (exposed to copper) conditions 166, 209. Using 300 mM borate BGE, pH 7.6, in bare FS capillary (75 μm id, 40/30 cm) and 25 kV separation voltages the separation of GSH and GSSG was achieved within 3 min.…”