If the reaction is divalent metal ion-dependent, you can stop the reaction by adding an excess of EDTA to bind the metal.

You can kill the protein by adding perchloric acid, then precipitate the perchlorate with an equivalent amount of potassium carbonate, leaving nothing but carbonic acid in the supernatant, which bubbles off as carbon dioxide. Centrifuge the sample and take the supernatant to analyze for the product.

A rather slow method is ultrafiltration, using a micro-centrifugal ultrafilter (e.g. Microcon) with a molecular weigh cutoff smaller than the molecular weight of the enzyme. This can be done at 4oC to slow down the reaction during the separation.

We have stopped the enzyme reaction by adding methanol (please see files; BPAB HPLC-BIN assay, Yamauchi BAQ, and Dr. Terentyeva Urine BIN) and methanol-acetonitrile (AN) solution (50 : 50 (v/v): please see file; J Chrom B rat BIN LIP Km).   Methanol-AN method seems to be better, since the volume changes due to evaporation are reduced.

This stopping method is uniquely applicable onto the reliable HPLC-enzyme assay method.

By the way, mess-up problemn is not present in the sensitive HPLC-enzyme assay method.   Therefore, you should develop an HPLC-enzyme assay method by yourself, since traditional photometric-enzyme assay method (without purification of enzymic product and substrate molecules) is not quantitative due to violation against the "Lambert-Beer's Law" giving bend calibration curve.   

Hi Sammy, in addition to the excellent answers above, you could try adding high concentrations of a competitive inhibitor to your enzyme solution.

It would be even better if you had a covalent inhibitor of your enzyme.

Enzyme active site directed inhibitors are extremely helpful in enzyme inactivation when traditional enzyme or protein denaturating approach does not work.

Low and very low  temperatures may be helpful to stop the assay if the reaction is sensitive to temperature. It is easy to settle : ice & water bath. If available liquid nitrogen can work too.

Anything that brings the pH out of the active range for your enzyme should work. For example, our enzymes are active in the pH range of 3.5 to 7.5, so addition of sodium carbonate to bring the pH to about 8.5 works well for us.

NaN3

You can also "crash" the protein by adding acetonitrile.

Understanding or having an idea of the enzyme in question will go a long way in getting the right inhibitor for the enzyme. however, a competitive inhibitor must be used and also the concentration of inhibitor must be greater than the concentration of substrate to prevent mass action effect.

Also, freeze clamping in liquid nitrogen can instantly halt enzyme activity and is used in kinetic studies for rapid result.

It must depends of all conditions of the reaction: stability of substrates, products and of course, the nature of the enzyme. In my experience I used Na2CO3 - 0,200 M to stop the reaction by denaturing the enzyme, but this solution is so alkaline, so you have to take this into account (it may react with the products of substrates). Another option is the addition of organic solvent to denature the enzyme, for example acetone of methanol. In both cases, I suggest to use the organic solvent at -20°C in order to precipitate the protein and quench the reaction as son as possible.

You could look for a specific inhibitor in the enzyme database BRENDA (http://www.brenda-enzymes.info/).

In addition to the general inhibitors acid, base and organic solvent discussed by others you could also use detergent, e.g., SDS. Some enzymes are inhibited by heavy metals, others by removing metal ions with chelators.

I don’t know if anyone has mentioned this, but you can filter the assay using a nylon membrane. It worked well for me. You can get these syringeless filters from whatman that have a nylon membrane and it’s pretty easy if you dont have a ton of samples

Exposing enzyme to low temperature and p H changes maybe helpful.