Dear all, I used to avoid air oxidation of GSH by homogenization in an inert atmosphere (nitrogen air flow) but have since changed labs. Most protocols simply add EDTA to the samples to avoid bivalent metal binding, but what do you do with your samples, or do you not worry about this at all? This would be for subsequent GSH determination by Ellman´s assay with DTNB only, not the enyzmatic method that uses glutathione reductase.
Thanks in advance,
Rachel
GSH autooxidation can be prevented by the use of N-ethylmaleimide (NEM). Please check this paper for more details: http://www.clinchem.org/content/33/8/1448.full.pdf
Regards, Christian
Dear Rachel although it is impossible to completely eliminate auto oxidation especially most derivatization procedures for determination of glutathione require high pH where auto oxidation is high,however with proper precautions the amount of auto oxidation can be limited .we usually promptly blocked GSH autoxidation in sample by adding N-ethylmaleimide (NEM) at 10mmole concentration and 17.5 mole of EDTA to avoid metals binding and used low pH buffer less than 7 ( For example potassium phosphate pH 6.5) under these conditions auto oxidation should be minimum .Good luck
Be careful: If you use derivatize reduced glutathione with N-ethylmaleimide (NEM), the GS-NEM conjugate will not react with 5,5′-Dithiobis(2-nitrobenzoic acid). The –SH will not be available to react with DTNB.
One alternative is to gently homogenize your sample in N2 bubbled acid solution (e.g. trichloroacetic acid or sulfosalicylic acid) in low temperature. This way you precipitate proteins and keep a low pH.
Thank you all for your answers, I will add EDTA and use a buffer at 6.5 (I currently use 7.0 PBS), unfortunately I don't have NEM at the lab right now, and have no N2 source at the moment, but I'll search for these at my institution. Another question: I see different protocols using 17.5 mM EDTA, 100 mM EDTA, 200 mM EDTA, 1 mM EDTA. The EDTA should simply be in excess, correct? So what would be the best concentration/amount to add? I usually work with 100 mg freeze-dried samples in about 500/600 uL PBS.
Hello Aurea, yes, unfortunately I dissect my animals, fish, rats, mussels... the dolphins and whales are by-caught by another research group, so they did all the dissecting, but yes, it's awful. I'm trying not to work with rats anymore, I used to cry when I dissected them, and I had to kill them by decapitation, awful method. Now I'm working with marine mammals, some fish and humans (blood and urine), so I'm better of!
I'll try 1 mM EDTA then! :D
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