Enzyme react with substrate and it gives some product which is detected based on its colour. For inhibitory assay, if am choosing coloured sample as test, which gives more colour at the end of assay mixture, how can we calculate the inhibitory %? Because test is more colour than control.
A method for correcting for interfering substances in assays is given in this paper:
http://jbx.sagepub.com/content/14/8/1008.long
In your case, set up a second set of samples with the same inhibitor concentrations but no enzyme to measure the absorbance caused by the inhibitor and subtract it from the absorbance measured with the same concentration of inhibitor + enzyme.
The limit of this method is reached when the inhibitor absorbance is several times higher than the absorbance due to the enzyme activity. However, by using the averages of triplicate measurements, you should be able to correct for inhibitor absorbances up to about 3 times the absorbance without inhibitor.
In the worst cases, it may be necessary to find a different assay method, preferably one that separates the inhibitor from the product (e.g. HPLC, LC-MS).
In simple you have to keep a blank (substrate free) but have all parameters same. And at the end you have to minus its OD (Absorbance) from your reaction mixture OD.
All the best.
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