Chromogenic reactions are those that result in formation of a chromophore at sites of enzyme activities. The great majority of these reactions were adopted from well-tried histochemical or colorimetric enzyme assay methods. In a simple one-step chro-mogenic reaction a colorless substrate is enzymatically converted into a colored product. In a broad sense, the class of chromogenic reactions includes any reactions or set of reactions that reveal discrete zones (or bands) of enzyme activity visible at daylight. In most cases the primary product(s) of an enzyme reaction is not readily detectable, and a supplementary reagent(s) is added to the reaction mixture, which somehow reacts with the primary product(s) to form a visible secondary product(s). These secondary products can be formed as the result of spontaneous reactions or so-called chemical coupling. In some cases, however, none of the primary products can be detected by chemical coupling, and an additional enzymatic reaction(s) is needed in order to reach a detectable product(s). This procedure is called enzymatic coupling, and supplementary exogenous enzymes that are added to reaction mixtures are known as linking or auxiliary enzymes. The resultant zymogram is positively stained (colored bands on achromatic background) if the color is formed by a reaction with the product of the enzyme activity. A negatively stained zymogram (achromatic bands on colored background) is obtained if the color is formed by a reaction with the substrate.
Many different enzymes produce the same molecules (e.g., NADH, NADPH, orthophosphate, ammonia, hydrogen peroxide, etc.) or different molecules with essentially the same chemical properties (e.g., aldehydes, ketones, reducing sugars, thiols, etc.). This means that very similar or identical chromogenic reactions can be used to detect different enzymes. Thus, classification of chromogenic reactions based on properties of products that are detected seems to be the more useful and practical approach. Such classification is advantageous because it allows one to choose an adequate chromogenic reaction to visualize activity bands even of those enzymes that have not yet been detected on electrophoretic gels.
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