are laboratory methods for measuring enzymatic activity.
Types of assay
All enzyme assays measure either the consumption of substrate or production of product over time. This gives the rate of reaction. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways.
In general, enzyme assays can be split into two groups continuous assays
, where the assay gives a continuous reading of activity, and discontinuous assays
, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.
Continuous assays are generally preferred since they are far more convenient, with one assay giving the rate of reaction with no further work necessary.
In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, these are called colorimetric assays. The MTT assay, a redox assay using a tetrazolium dye as s substrate is an example of a colorimetric assay.
UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do not in their oxidised forms. A oxidoreductase using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at 340 nm as it consumes the coenzyme.
Direct versus coupled assays'
Fluorometric assays use a difference in the fluorescence
of substrate from product to measure the enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interferance caused by impurities and the instability of many fluorescent compounds to light.
An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase.
[Passonneau, J. V., and Lowry, O. H. "Enzymatic Analysis. A Practical Guide", Humana Press (Totowa, NJ:1993), pp.85-110.]
Synthetic substrates that release a fluorescent dye in an enzyme-catalyzed reaction are also avalible, such as 4-methylumbelliferyl-β-D-glucuronide for assaying β-galactosidase.
Factors to control in assays
- Temperature Most chemical reactions speed up as temperature is raised. Extremes of temperature can denature an enzyme so that it can no longer function. The temperature at which the enzyme exhibits maximum activity is called the enzyme's temperature.
- Substrate concentration
- Enzyme concentration The rate of an enzyme reaction is directly proportional to enzyme concentration.
- pH Extreme of pH can denature an enzyme so that it can no longer function. Many enzymes function optimally around the neutral pH region in pH 5-8. However, there are exceptions; pepsin, a stomach enzyme, functions only in very acidic conditions, and so cannot work in the small intestine.
- Salt concentration Extremes of salt concentration can inactivate an enzyme. Typical enzymes are active in salt concentrations of 1-500 mM.
List of Enzyme Assays