I'm studying heavy metal toxicity in wheat. I need to prepare a standard for GSH measurement, and I could not determine the appropriate GSH standards. Does anyone have any information?
Hello Betülnur Özel
We use L-CYSTEINE as standar with great results. Any brand is suitable.
We use the Ellman assay.
Good luck
Only HPLC assays (with fluorimetric detection) enable you to use an internal standard which should then be distinct from GSH.
If you are not using chromatography, but instead a direct colorimetric or fluorimetric assay of GSH, you will use an external standard and the best one is GSH itself to get a standard curve.
The most important point is that aqueous solutions of GSH are not very stable (true also for cysteine and most other mercaptans) because they easily get oxidized (GSH oxidized to GSSG) above pH 5, and more rapidly with increasing pH. At biological pH (typically between pH 6 and 8), unacceptable loss of GSH may be observed within 1-3 hours because of oxidation, and if you are dealing with biological samples, catalytic degradation of GSH will worsen the situation (gamma-glutamyltranspeptidase and glutathione oxidase activities are the main problems). So stabilizing your standard solutions and your biological extracts in acidic medium is mandatory.
It is usually recommended to prepare and store your standard solutions in 5% metaphosphoric acid (especially if you want to measure both GSH and GSSG), but metaphosphoric acid is itself not very stable. For rapid use (within 24h), my experience is that 10 mM acetate buffer pH 4.7 works well to assay GSH (GSH only) with most existing methods. Be careful however to check that this buffer will be sufficiently diluted in the final reactional medium to avoid interference with the pH required in your assay protocol.
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