Editing Isozyme (section)

==Distinguishing isozymes ==
Isozymes (and allozymes) are variants of the same enzyme. Unless they are identical in their biochemical properties, for example their [[Substrate (biochemistry)|substrates]] and [[enzyme kinetics]], they may be distinguished by a [[biochemical assay]]. However, such differences are usually subtle, particularly between ''allozymes'' which are often  [[Neutral theory of molecular evolution|neutral variants]]. This subtlety is to be expected, because two enzymes that differ significantly in their function are unlikely to have been identified as ''isozymes''.

While isozymes may be almost identical in function, they may differ in other ways. In particular, [[amino acid]] substitutions that change the [[electric charge]] of the enzyme are simple to identify by [[gel electrophoresis]], and this forms the basis for the use of isozymes as [[molecular marker]]s. To identify isozymes, a crude protein extract is made by grinding animal or plant tissue with an extraction buffer, and the components of extract are separated according to their charge by gel electrophoresis. Historically, this has usually been done using gels made from [[potato starch]], but [[acrylamide]] gels provide better resolution.

All the proteins from the tissue are present in the gel, so that individual enzymes must be identified using an assay that links their function to a staining reaction. For example, detection can be based on the localised [[precipitation (chemistry)|precipitation]] of soluble indicator [[dye]]s such as [[tetrazolium salts]] which become insoluble when they are [[Redox|reduced]] by [[Cofactor (biochemistry)|cofactors]] such as [[Nicotinamide adenine dinucleotide|NAD]] or [[NADP]], which generated in zones of enzyme activity. This assay method requires that the enzymes are still functional after separation ([[native gel electrophoresis]]), and provides the greatest challenge to using isozymes as a laboratory technique.

Isoenzymes differ in kinetics (they have different [[Michaelis-Menten kinetics|''K''<sub>M</sub> and V<sub>max</sub>]] values).