Enzyme activity can be measured by measuring the concentration of product of concentration of disappearing substrate but measuring concentration of product will give better idea about the enzyme activity.

Every enzyme has its specific substrate and product one can make standard curve of the product with reference of that standard curve concentration of product and with Michaelis-Menten Kinetics enzyme activity can be calculated.

you can make a molecule that enhances or inhibits the activity of the enzyme that you can try to modulate the activity of the genetic network rather than impinging upon the product.lipid-soluble reaction product is formed by catalyzing an addition reaction of two or more kinds of substrates, characterized by comprising a labeling step in which any one of the substrates is labeled, a reaction step in which an enzymatic reaction is carried out in the presence of an enzyme, all substrates and an acceptor, and a detection step in which the reaction product is detected by bringing the labeled molecule and a molecule to be detected close to each other via the acceptor and transferring an energy generated by the labeled molecule to the molecule to be detected, and a method of evaluating a compound utilizing the method of measuring an activity. By the methods of the invention, it is possible to perform a treatment from the enzymatic reaction to the measurement of an enzyme activity with the simple steps and it is possible to provide a method of measuring an enzyme activity which can be applied to an HTS evaluation system.

For your first question, I suggest to you the following important reference for food enzymology: "Enzymic Hydrolysis of Food Proteins" by Jens Adler-Nissen (1986).

Is there a known substrate for the enzyme, e.g. from Sigma (you should exactly know where the enzyme cleaves)? So you could perform a colorimetric assay. Use a substrate (or better several substrates for comparisons), that might be cleaved by the enzyme of interest and measure the colorimentric reaction using ELISA reader at the indicated wavelength (be aware of that e.g. phenol red (at 406 nm) and other agents can contribute to the measurement as well).

Additionally, you should create a standard curve with an enzyme of known concentration. Then you can see more or less how active your enzyme is.

Important is to use adequate buffers and concentrations. Sera contain enzyme inhibitors. PM for further advise. Thank you.

To determine enzyme activity against a compound you should know well about what substrate for enzyme and what optimal condition (pH, temperature, concentration of and enzyme and substrate, buffer supported for the reaction, reaction time). If you know what enzyme do you use, you can find exactly other information on sigma, and so many reference paper. After that, Using spectophotometer or ELISA reader to determine concentration of the compound are produced after reaction of enzyme and substrate. From that, you can calculate the enzyme activity or enzyme inhibitory activity of target compound.

For the secondary question: In case using ELISA reader to determine enzyme activity as I mentioned before; That is totally difference with radioactive assay.

Good luck

An ELISA measures total protein or chemicals such as steroids, etc, by immunologically detection. So anything that is used by an enzyme whether it be substrate or product or the enzyme itself, can be detected with an ELISA. An ELISA depends on the binding of a secondary antibody, to the primary antibody. The secondary antibody can have a detection enzyme attached like HRP. The HRP can be measured colorimetrically, fluorometrically, or with chemiluminescence. A radioimmune assay is different. It uses a radioactively tagged Antibody as a secondary antibody.

For ezyme assays, the decrease in substrate or increase in product and the amounts of each can be measured by ELISA, RIA, or other techniques. The method needed depends on the enzyme, and how you want to detect substrate to product conversion. RADioactive, colorimetric, and fluorometric assays that are not based on antibody detection are most often used with enzymes. The radioactive assays typically have a substrate labelled with C13, H3, P32, and separation of substrate from product with HPLC, or other simple techniques, like binding to beads or supports based on charge differences or size differences.

Other assays, based on color, fluorescence or chemiluminescence, typically work by producing a signal when the substrate is converted to product. One example is paranitrophenyl phosphate conversion by a phosphatase to paranitro phenol. When the phosphate group is released by the phosphatase, the reaction turns yellow due to the production of paranitrophenol.

I forgot to add that if using an enzyme, a compound can either increase, decrease or not affect conversion of substrate to product. This means that whatever type of assay you use, you will have to measure substrate conversion to product.

Thank you all for your valuable comments.