Which method is best for GCL assay and Why?

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  • #1
TytoAlba95
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I'm trying to choose a protocol for estimating GLutamate Cysteine ligase assay. I've two of them.
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L-glutamate + L-cysteine + ATP
\rightleftharpoons
gamma-glutamyl cysteine + ADP + Pi

#Protocol 1: Dasgupta 2007
Though this method, the author has estimated GCL activity by measuring a blue coloured compound formed by a reaction between Pi and Ferrous sulphate-ammonium molybdate reacgent. She has basicially estimated Phosphate.

#Protocol 2: Seelig 1985
It is a coupled enzyme assay.
The enzyme activity is measured in reaction mixtures containing L-glutamate, L-a-aminobutyrate, and ATP by a coupled enzyme procedure in which the rate of formation of ADP, in the presence of pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, and NADH, is obtained from the decrease in the absorbance of NADH at 340 nm.

Which one of these is better and why?
 

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  • #2
TytoAlba95
132
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My take on :
Which one of these is better and why?

I think doing the double coupled enzyme assay is better over estimating the inorganic phosphate. Because inorganic phosphate is rather ubiquitous and the coupled enzyme assay will estimate the decrease in NADH which is supplied from outside and added to the basal level of NADH present in the tissue extract, which can be substracted.

#Here's a link (Taussky 1953) to the original paper from which Protocol 1 is derived.
 

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