I am performing glucose 6-phosphate dehydrogenase assays using a spectrophotometric method but I am unable to obtain the expected activity from the stock enzymes. My suspicion is that the NAD is damaged as there seems to be a correlation between the specific reaction rate and NAD concentration, despite the NAD concentration being substantially greater than Km.
Hi Prasoona & Muhammad,
Thanks for the answers.
I am doing the assay at 37 C and pH 7.4 following the Sigma-Aldrich method (see link), although at a higher temperature. Since I am monitoring the conversion of NAD+ to NADH, I am working at a wavelength of 340 nm. The enzyme converts glucose 6-phosphate to 6-phosphogluconolactone using NAD+ as cofactor.
What is the shelf life of NAD stored as aliquots at -20 C and what is the most suitable buffer and concentration for storage?
Thanks,
Michael
http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-glucose-6-phosphate-dehydrogenase.html
How much higher above the Km are you. The reaction rate will increase somewhat even above the Km. But if you are 10 fold higher, or above, then the NAD+ concentration is probably not what you think, perhaps because of degradation.
Thanks for the advice Prasoona.
Regarding NAD+ concentrations, I have tried a range of values from 9x to 200x the Km value but still do not observe the expected enzyme activity, however there appears to be an increase in activity with increasing NAD+ concentration. There does not appear to be a similar correlation for G6P. The Km values I have found in literature are 0.11 mM and 0.02 mM for NAD+ and G6P respectively.
It may be that the stock enzymes and the NAD+ have degraded, although I have tested two batches of each.
Try checking the concentration of the NAD stock solution using its UV absorbance.
You should try different concentration of your enzyme from nm to mg. These kind of essays are very sensitive to the enzyme concentration, if is too little or too much you can't see anything. Is it purified? try also 30 degrees... Another thing, NAD should be the last put in your reaction and immediately measured over continuous measurement for at least 10 min.
NAD solutions stored at about -20° are quite stable for at least a month but keeping it in a modestly neutral pH with buffer solution is also a good practice. With the assay you describe, the concentration can easily and should be well above the Km value so that this reaction is not rate limiting.
Checking stability and concentration via UV is a very good suggestion and to check this over time to see if that is your problem (and it should not be) is also a good suggestion.
G6PDH uses NADP+ and G6P as a substrate in most organisms, but some Organisms' G6PDH can us both NADP and NAD. Try testing the reaction with NADP to make sure the NAD is the problem and not the experimental design.
Thanks for all the useful tips. I'm leaning towards the cofactor specificity being the problem, will try get my hands on some NADP+ to confirm. Also, UV measurements (259 nm) of NAD+ solutions seem to indicate that the actual concentration is substantially lower than the expected concentration. How reliable are measurements of NAD+ at 259 nm?
Higher concentrations tend to deviate from the Beer-Lambert law. It would be useful to measure a variety of concentrations and look at the percentage of maximal absorbance. NADH at 340nm in a 96 well plate, 100ul, for example, deviates quite a bit at concentrations higher than 1mM
It turns out there are three classes of G6PDH. One class has specificity for NADP only (the stock enzyme I was testing), while the other two classes have varying degrees of specificity for NADP and NAD. The surest approach is to use NADP since all classes can readily use this as the coenzyme. Thanks for the helpful responses.
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