For enzyme assays using cell supernatant which are slight yellow and using pnpp substrate which will also produce the yellow pnp substrate will predictably have a distortion on the absorbance. to what extent will this affect enzyme rate calculation and any papers on this?
Hi there,
Make the zero of the spec by using the complete reaction mixture lacking pNPP (ie. including the cell supernatant ).
Apply blank subtraction for each sample individually by including both spiked and nonspiked sample and measure thr absorbancr difference between the read...It may be better if you can stop the reaction by shifting pH to inactivating condition for sample blank...after quenching spike the pnp...
You can subtract a blank, as already stated, but it isn't really necessary as long as you are sure that the background absorbance due to (1) the supernatant without pNPP and (2) pNPP without the supernatant does not change over the time course of the full reaction. That is because you only need to know the rate of change of the absorbance caused by the enzyme-catalyzed reaction to measure the activity.
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