Determination of Glutathione and Glutathione Disulphide in Lichens: a Comparison of Frequently Used Methods

✍ Scribed by Ilse Kranner; Dieter Grill

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 575 KB
• Volume: 7
• Category: Article
• ISSN: 0958-0344
• DOI: 10.1002/(sici)1099-1565(199601)7:1<24::aid-pca277>3.0.co;2-2

✦ Synopsis

The determination of glutathione (GSH) and glutathione disulphide (GSSG) is subject to substantial problems when analysing plants containing high amounts of phenolic compounds, since the phenolics may react with the GSH present during the extraction. Three frequently used methods for the analysis of GSH and GSSG in the lichen Pseudevernia furfuracea have been compared. Glutathione seemed to be underestimated by the enzymatic recycling assay. Determination by high-performance liquid chromatography after derivatization of glutathione with 2,4-dinitro-l-fluorobenzene (DNFB) or labelling with monobromobimane (mBBr), respectively, gave similar results. Derivatization with DNFB gave the lowest values for GSSG expressed as a percentage of total glutathione. Cyst(e)ine and y-glutamyl-cyst(e)ine were only detected with the mBBr assay.

In order to improve the yield of GSH, polyvinylpolypyrrolidone (PVP), which should serve to bind phenolics, was added during the extraction. The use of PVP improved the yield of glutathione dramatically in lichens harvested in June but not in October. However, addition of PVP led to oxidation of a portion of the GSH. This was demonstrated for each method and seemed to be independent of the thiol blocking agents used for the determination of GSSG, namely 2-vinylpyridine, iodoacetic acid or N-ethylmaleimide. For extensive studies on glutathione metabolism in Pseudevernia furfuracea we recommend that the use of PVP be avoided, by investigating thalli collected in autumn, that probably contain less phenolics than those harvested in summer.