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

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.