Hi Abdelmageed,

   Whenever you perform an enzyme assay, you monitor for the conversion of substrate to product that is reported as an increase in absorbance (or decrease in absorbance depending on the way the assay is set up). The slope of this line reports the change in optical density value per unit time. You can use the beer-lambert's law to compute the concentration of substrate consumed/product formed from the change in optical density as follows

Absorbance= epsilon*concentration*pathlength

Since you know the absorbance (dimensionless) from your curve and the pathlength (dimensions of length) from your experimental setup, and since epsilon is a constant for a particular (dimensions of concentrations inverse and length inverse) metabolite at a particular wavelength, you can compute the product formed/substrate consumed (dimensions of concentration) by rearranging the equation as follows

concentration =Absorbance/epsilon(concentration^-1length^-1)*pathlength(length)

I hope this answer helps!

Dear Abdelmageed,

As you might know that the amount of enzyme needed for converting a substrate to products is a catalytic amount. What matters is the concentration of the substrate and products.

If you follow the steps described by Bharath you will be able to calculate the amount or the concentration of the product/s.

Good luck,

Rafik

Thanks, Bharath Srinivasan. I think the method you stated is valid for the kinetic measurements, but I talk about using the standard curve of the product how I can convert  the units obtained to micro moles. Thanks

Hi Abdelmageed,

     The method explained by me is equally applicable to both end-point and continuous assay systems provide you have the delta extinction coefficient. However, if you have a standard curve than the calculation becomes all the more easy since you can compute the moles from your standard curve using the molecular weight of the compound and extrapolate the O.D obtained from your test case onto the standard curve. I hope this helps!

Thank you very much Bharath Srinivasan for your helpful and clear answer.