I think this is an excellent solution to the problem I have by Arne. Pasted here for the benefit of others that have similar problem.

"Hi

I understood the equation given in your link that if you follow it directly you don't have to care about the 3 ml/0.5 ml. The equation contains the term "0.001" which is the change in absorbance per time in a 3 ml reaction (so this takes the 3 ml into account). Additionally you have a term for the volume of added enzyme (0.5 ml), which normalizes your activity per volume. If you added more enzyme, let's say 1 ml, then the 0.5 ml needs to be changed to 1 ml and the 0.001 needs to be recalculated for a 3.5 ml reaction, instead of a 3 ml reaction.

The dilution factor (df) in this case is not 3 ml/0.5 ml because that has already been taken into account above. Only if you dilute your enzyme (let's say, 10 fold), then df needs to be 10, because in this case you observe only 1/10th of the activity of you undiluted enzyme.

Does this make sense? Hope so... :D

In principle the equation given on the webpage is the conventional equation, but it has been rearranged to contain a single combined term (the 0.001) for the extinction coefficient, pathlength and total reaction volume. This has probably been done for conveniency, and they assume that when you follow the steps on the web page you always only use this term. If you change any parameter (like pathlength), then obviously this term would not be 0.001 but something else, and needs to be recalculated."

-Arne

Many people prefer the ABTS assay.

Yes Shelley, I am using the ABTS assay as well.

Thanks!