Good evening,
Could somebody please send me a spectrophotometric determination method of GSH and GPx !
Thank you
This is an old method (spectro) using in my previous lab.
GSH was assayed by the enzymatic recycling procedure in which it is sequentially oxidized by 5,5- dithiobis (2-nitrobenzoic acid) and reduced by NADPH in the presence of GR according to Griffith (1980) and modified by Lutts et al. (2004). Briefly, extraction was performed in 5% sulphosalicylic acid on ca. 1 g fresh weight (FW) ground tissue and the extent of 2-nitro-5- thiobenzoic acid formation was monitored at 412 nm for GSH plus GSSG evaluation. For determination of GSSG alone, the extract was pre-treated with 2-vinylpyridine to scavenge GSH by derivatization.
Another suggestion is using HPLC. You can read my paper:
NaCl impact on Kosteletzkya pentacarpos seedlings simultaneously exposed to cadmium and zinc toxicities
Hopefully, it is useful for you
For example in the serum, Glutathion is determined by a modified procedure utilizing Ellman`s reagent (Sedlak and Lindsay, 1968). In brief; an equal volume (150 µl) of serum and 4% sulfosalicylic acid were mixed, centrifuged (314 x g ) at 4 ºC for 5 minutes.
To 150 µl of supernatant, 4.5ml of 0.1 mmol Ellman`s reagent (5,5-dithiobis(2-Nitrobenzoic acid DTNB) was added in phosphate buffer of pH 8.0 (Prepared by mixing of 0.6 mol KH2PO4 and 0.08 mol K2HPO4) , then the absorbance at 412 nm was measured.
Sedlak, J. , Lindsay, R.H.(1968).Anal. Biochem.192.
Sameh Boukerche
For GPx you can use my search below:
Article Partial Purification and Some Kinetic studies of Glutathione...
Luay A. Al-Helaly thank you for responding. I found the original article of Ellman (1959) and the moddified one, by Sedlak (1968) too. BUT I have some confusing pointes.
OK ... but it is a good and easy method that has been used in many of my research...
Sameh Boukerche
Asmaa A.khalifa Thank you so much, it did helped me a lot. I just have a question; where did you get the calculations, i didn't find them on any article even the originals ! and should i do the calibration range in every procedure ? Thank you for your answer.
@sameh
The equations are related to each procedure so you can find it in any article using colorimetric method you can find a citation for it
Standard curve also mentioned for some parameters
Some methods required only the application of the equation for each sample
In case you want to work with a kit data for standard curve drawing will be available
Asmaa A.khalifa could you please send me the article that you worked with for the determination of Glutathione peroxidase, i couldn't find it , thank you so much
Please check this article as a good example for using the GPx determination method with citation of the original method introduced in 1970s
Hope you got it , good luck dear
You can also google the method by using its name you will find more article
@Asmaa pls could you provide the reference to the protocol on glutathione. I used it but needed to reference
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