I am working on Nitrocefin assay for screening of beta-lactamase inhibitors and for that I would need to generate the standard curve using Nitrocefin. For my assay, I would be using crude beta-lactamase enzyme and inhibitor which will be incubated and later Nitrocefin will be added and evaluated using microplate reader at 490nm. Can I plot standard curve using Nitrocefin (fixed concentration) with enzyme unit (varying concentrations) or Is there any other methodology ? Please suggest as I am not using any readymade kit and need to develop a simple working procedure in a lab.
The enzyme activity is quantified by the initial rate of the absorbance increase (the slope of the A490 versus time curve at the start). To convert this into the rate of nitrocefin hydrolysis (µmoles/min), you need to measure the extinction coefficient difference at 490 nm between nitrocefin and its product.
Since you don't have the product, you can make it in situ by mixing various concentrations of nitrocefin with enough of the enzyme to completely convert it to product in a reasonable amount of time, subtracting any background from the same concentration of enzyme without nitrocefin (since it is an impure extract). Plot the absorbance at the endpoint versus the concentration. The slope of the plot is the extinction coefficient difference (Delta A490/µmolar).
Once you have that number you can convert the A490/min initial rate to µmolar/min by dividing by the extinction coefficient difference (A490/µmolar). Multiply by the reaction volume in liters to get µmole/min.
A nice advantage of this method is that it automatically accounts for the geometry of the measurement, i.e., whether it is in a cuvette in a spectrophotometer or a multiwell plate in a plate reader, as long as the standard curve is prepared under the same exact conditions as those in which the beta-lactamase assay is run.
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