formula for enzyme units

[ash]

what is exact formula to calculate units of enzyme activity from reactin during enzyme assay. if one would want to determine it by HPLC method, is this formula same for both spectrophotometric and HPLC assay.

with regards
ash

[Terry]

Alot of it depends on the method and how the unit is defined.
Another assumption is substrate in xs
At least for the ones I have run.
...no flip flops.

Methods are sometimes written as

a. A relative method...one requiring a standard which has a declared value as XXX Units/gm or XXX Units/ml or something like that.

or

b. An absolute method is one with no standard but the products are determined quantitatively. If you are lucky, your products can be direct read someway, but usually you may have to put them through further reactions to get something readable. In this case your calculations will depend on the definition of the unit.

If you substitute a HPLC "finish" for a Spectrophotometric "finish" using a releative method then you have treated standards along the way and it can be as simple as regressing from a line (or whatever calibration model is used) back to your unknown.

If you substitute a HPLC "finish" for a Spectrophotometric "finish" using a absolute method then you must rely on the definition of the unit.
Unit defintions are usually described as moles of product formed from a particular subtrate at a given temperature at a given pH per time. Then you calibrate the HPLC with product, determine mole of product..correct for reaction time and so on.
I have run many absolute methods using the molar extinctions of products for spectrophotometic assays, and they usually can migrate over to HPLC if the reaction solutions are terminated and stable.

It can get a bit hairy, and this is an oversimplication because these assays have to be concerned with interferences, backgrounds, blanks and so on.

That's my 0.02... and I guess the particulars would depend on the specific enzyme you are interested in.

Hope that helps a bit.
Terry

[ash]

hi
Thanks for your sugession
wiht regards
ash