For my experiment, I am running an enzymatic reaction, if I need to quantify the product but I don't have a standard or extinction coefficient at a specific absorption. Can I use the decrease in substrate to calculate the initial velocity of my reaction?
To do that you have to perform standard addition of the substrate. But the amount of product cannot be estimated unless a reaction mechanism is known and no by products are formed.
I have known the mechanism of the reaction but there is two products the first one is an intermediate and it is not stable to prepare a standard curve for!
I don't have a method for determining the rates spectrophotometrically, because I use very low concentrations of the substrate. I work with monooxygenase that has FAD reduced first by NADPH and then oxidize the substrate which is an antibiotic. the products are very similar to the substrate (analogues) and behaves nearly the same with most of the reagents. so I couldn't find a way to calculate just one component without having interference of the other.
For HPLC, I am not sure I have got What do you mean Dr. Anwar. , Won't dioxane affect the enzymatic reaction. as the intermediate is not converted spontaneously into the the final product, it is an enzymatic process! . if the first product doesn't converted by the enzyme it will be degraded to a different compound I can only detect its signal with HPLC.
Dear Heba
If you are going for Quantification you must require standard as well as IS by HPLC.
As you asking
"decrease in substrate to calculate the initial velocity of your reaction"
It depends the behaviour of your substrate. From it you can define the corresponding constant factor of rate of your reaction not the quantification.
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