The Problem of Glutathione Determination: a Comparative Study on the Measurement of Glutathione from Plant Cells

Abstract

The level and redox status of glutathione is a good indicator for the rate of oxidative stress and eco-toxicological injury in plant cells and subcellular organelles. Thus the determination of GSH and its redox status has special importance. A variety of spectrophotometric and HPLC methods are available to measure glutathione (GSH). The spectrophotometric DTNB-GSH recycling assay is specific due to the application of glutathione reductase, it is rather quick and easy to perform, not surprising that it is rather popular. In the present study we make an attempt to compare the DTNB-GSH recycling assay and the more sophisticated, but difficult monochlorobimane (mBCl)-HPLC method to choose the one that best suits for eco-toxicological and plant stress investigations. We found that the acidification by sulphosalicylic acid (SSA) used for the stabilization of samples for DTNB-GSH recycling assay gives lower efficiency to this method than the formation of mBCl-GSH fluorescent conjugate. The measurable GSH contents were lower in the case of DTNB-GSH recycling assay than in the case of GSH-mBCl conjugates determined by HPLC with fluorescence detection. The auto-oxidation could almost perfectly be prevented by the presence of mBCl in the organelle isolation buffer. Furthermore, this way the reduced GSH content of organelles could be determined much more precisely. However, it is worth to note that the application of mBCl significantly elevates the cost of GSH determination, especially in case of cell organelles.

Analytical methods for determination of glutathione and glutathione disulfide in pharmaceuticals and biological fluids

“…The reported normal levels of GSH and GSSG in human plasma are highly variable, which could be due to possible artifacts in measurement procedures (Rossi et al, 2002;Hajdinák et al, 2019). Accurate determination of GSH/GSSG in biological samples requires precautions during collection, storage, preparation, and analysis.…”

Genetic Polymorphism of GSTP-1 Affects Cyclophosphamide Treatment of Autoimmune Diseases

“…The GSH content of the samples was conjugated immediately by monochlorobimane (1 mM) in the presence of glutathione-S-transferase enzyme (100 mU/mL). The reaction was carried out in the dark at room temperature for 15 min and stopped with the addition of trichloroacetic acid at a final concentration of 10% (w/v) [41,42]. The samples were centrifuged at 16,000× g, at 4 • C for 10 min to pellet the precipitated proteins.…”

“…Standards were prepared from reduced glutathione dissolved in 5% SSA [0-20 µM], and their glutathione content (similarly to the samples) was conjugated with monochlorobimane. Based on our standards, the retention time of glutathione conjugated by monochorobimane was 8.5 min [42].…”

Determination of Glutathione in Austrian Wine Samples: The Effects of Freezing, the Choice of Yeast and Storage

“…A simple mechanism of protection is based on a reaction in which the reduced glutathione form (GSH) is oxidized to glutathione disulfide (GSSG), thereby releasing protons and electrons used in coupled reactions to preserve molecules against oxidation [11]. Additionally, the level and redox status of GSH in plant can estimate the rate of oxidative stress and eco-toxicological injury [12].…”