I am working on textile dye degradation by bacteria and want to calculate the azoreductase activity (IU/ml or IU/mg protein) from the absorbance value and extinction co-efficient value of NADH.
Thanks for sharing your experience.
Hi there,
As you are following the consumption of NADH at 340nm, initiao OD value has to be within the linearity domain of reading of your spec (usually between 1 and 2). Then other assay mixture components have to be set in order to observe a linear decrease of absorbance (initial velocity). Basically enzyme has to be far less than substrates. The slope is giving you the rate of NADH consumption. Theen you may convert it into molarity using NADH epsilon and then into mole number knowing the volume of the assay. If you know the mass of enzyme used then you may calculate its activity in mol/min/mg and then convert into IU (1IU being 1µmol/min). For further info, you might be interested in reading one of my papers where azoreductase assays are run...
https://www.researchgate.net/publication/8523402_Crystal_Structure_and_Functional_Characterization_of_Yeast_YLR011wp_an_Enzyme_with_NADPH-FMN_and_Ferric_Iron_Reductase_Activities
Article Crystal Structure and Functional Characterization of Yeast Y...
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