Hello all, we'd like to pick your brain .
The enzyme we're testing requires NADPH to work. We have controls showing that our reaction needs our enzyme and our enzyme definitely needs NADPH. Since NADPH is quite pricy, we have been adding glucose, GDH and NADP+ to form a NADPH/NADP+ cycle. Interestingly, we found that the activity of our enzyme is at its highest when we only add GDH and glucose. We lowered the NADP+ concentration. The enzyme activity is higher than the original reaction but still not as high as when no NADP+. We thought maybe the NADP+ we added is too much which could suppress our reaction. We are wondering if anyone has seen something like this before and what do you think of our hypothesis. Thanks!
Hi there,
NADP+ being one of the products of the reaction you wish to measure, its addition (how much do you add?) encourages the phenomenon of inhibition of the reaction by one of the products. If the reaction works OK with glucose and GDH alone it means the internal pool of NADP+ is enough to make multiple turnovers. But if you want to control the concentration of NADPH available you would have to dialyze the enzyme extract first and then add NADPH.
Have you tested NAD+?
Often enzymes can use both NAD and NADP having higher affinity and reactivity with one or the other.
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