What kind of chemical agents can I use to oxidize NADPH in a reaction and stop an enzymatic reaction? Suggestions other than chemical agents are also welcome. Thanks!
Do you need to oxidize the NADPH, or is it sufficient just to stop the reaction? NADPH can be converted to NADP enzymatically by various commercially available dehydrogenase enzymes, combined with the appropriate substrate. Your enzyme reaction can be quenched by adding EDTA to chelate divalent metal ions if it is a metal-dependent reaction, or by acidification, or by adding a denatuant such as urea or SDS, or by adding a specific inhibitor if one is known.
Hi Adam. Actually this is related to another question I asked before on research gate. You also responded to that question - the one regarding two molecules absorbing at the same wavelength in an assay. The cofactor that I mentioned in that question is NADPH. I hope that there would be a way to quench the reaction by oxidizing NADPH so I could measure the absorbance increase from the substrate instead.
It should be possible to convert the NADPH to NADP enzymatically by adding a suitable enzyme and its substrate, being careful of course that the reaction does not produce another substance that interferes with your absorbance measurement. The enzyme diaphorase, for example, will use NADPH as a substrate, but you have to provide something else for it to reduce, and it is a flavoenzyme, so it has an absorbance of its own in the near UV. Also, conversion of NADPH to NADP does not completely eliminate its absorbance.
I have 2 suggestions:
(1) If you know that there is a stoichiometric (1:1) production of NADPH and the product you are interested in, then follow the NADPH production as a stand-in for the production of the other product. You can do this without interference from the absorbance of the other product by using diaphorase combined with the colorimetric substrate XTT to generate a product with a visible wavelength absorbance. The bonus is a nearly 4-fold increase in sensitivity. Or, another way to look at it is that since both the product of interest and the NADPH cause an increase in absorbance, and there is equal production of both of them, then you can simply follow the total change in absorbance. The change in absorbance is the sum of the changes in absorbance caused by the two products.
(2) Use a separation technique to measure the other product. HPLC or UPLC would be my suggestion, if the equipment is available.
Hi Adam. Thanks for the suggestions. I was going to use HPLC but I felt the run time (about 1 hour for each sample) would be too time consuming for obtaining kinetic data especially if each time point has to be done in triplicate. As for the first suggestion, the NADPH is being consumed in the reaction and so the absorbance its decreasing at 340 nm while the product
By spending some time Spending some time to optimize the HPLC conconditiin order to minimize the run time may be well worth it. It should not take afull hour per run. If there is a UPLC available, run times can be greatly reduced because of the higher resolution of the columns.
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