Pyruvate Determination - (Sep/17/2009 )
I have been attempting to setup a microplate assay for the determination of pyrvuate using the following reaction:
Pyruvate + NADH + H+ -----> Lactate + NAD+
I have tried using both absorbance and fluorescence detection but with no success. I was wondering if anyone had a good protocol or reference for a microplate based assay? I realise that I could just simply buy a kit, but I have already managed to setup microplate assays for several other metabolites with great success, but this one is proving to be a horn in my side.
All suggestions & help will be gratefully received,
Pyruvate is very unstable. I suspect in microtitre plates you will have very small volumes. Adding metaphosphoric acid may stabilise the pyr.
Does your colorimetric method (Abs at 340nm) work with NADH standards?
There is a gas chromatography method, first published in 1966. Not sure if any one uses it now, though. Savory and Kaplan (Clin Chem 12 559-569).
when running an assay that utilizes nad(h), i always followed the conversion of nad(h) over time with continuous monitoring at 340nm.
in your case, you would follow the conversion of nadh to nad. this reaction causes a drop in absorbance so blanking is a problem. if you blank with nadh then the reaction will go to negative absorbance. if you blank with nad then you can't run the reaction.
you will have to blank prior to the addition of nadh.
for a microplate assay you will have to take timed readings (unless you have a reader that can take continuous readings of all wells simultaneously), but, to do this you will have to be able to add the nadh to all of the wells simultaneously and then make timed readings.
as tedious as it was, i always sat at the spec and ran each assay, one at a time, preparing each one immediately prior to reading, and you may have to, as well (even if you found a way to stop the reaction without causing decomposition of the remaining nadh, you would still have to properly time the reactions because nadh is unstable).
(sorry about the rambling, run on sentences)