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Enzyme catalyzed colorimetric reactions (i.e. the use of enzymes as reagents in colorimetric reactions) are attractive because of the high specificity of enzymes for given substrates, the high specificity of products produced from the analytes and because of the impressive catalytic activity of enzymes.

Enzyme mediated reactions are among some of the most selective assays available for chemical measurements. Enzymes can readily distinguish between different constitutional isomers and different stereoisomers. For example, many sugar oxidases can oxidize only one type of sugar in a complex sugar mixture. Some of the enzymes involved in the degradation and synthesis of biopolymers have remarkable specificity and are used extensively in the characterization of long chain biopolymers. Examples of the uses of such enzymes are the use of PCR reaction in the amplification of specific DNA primers, the use of restriction enzymes in genetic mapping and the use of different amylases for the determination of starch in the presence of cellulose. (see figure 8.1).

Figure 8.1 A comparison of the reactivity of starch and cellulose

Starch = -D-(1--4) linkages of glucose-(glucose)n-glucose

Cellulose = -D-(1--4) linkages of glucose-(glucose)n-glucose

Acid Catalyzed Hydrolysis

Starch ----- Glucose

Cellulose ----- Glucose

Amyloglucosidase Catalyzed Hydrolysis

Amyloglucosidase
Starch ------------------------------ Glucose

Amyloglucosidase
Cellulose ------------------------------ Glucose
Not only are enzyme mediated reactions specific for the analytes , they are also quite specific in the products produced by these reactions. For example, while traditional non-enzymatic oxidations of glucose can produce a variety of thermodynamically possible products which they are further oxidized, the enzyme “glucose oxidase” quantitatively produces D-gluconic acid from D-glucose.
(see Figure 8.2)

Enzymes have remarkable catalytic ability and their use as catalysts generally increases the reaction rate of a given reaction somewhere between a 106 and a factor of 1020. Therefore, the use of enzymes as reagents in colorimetric reactions often significantly reduces the time needed for the analysis.

There are thousands of different enzymes. If an analyst needs to analyze for a compound that is found in nature, there are sure to be enzymes that synthesize the compound and others that degrade it. Chemical analyses of many compounds which are not found in nature can still be achieved with enzymes. There are many “synthetic substrates” for various enzymes, for example, p-nitrophenylphosphate is a synthetic compound which is hydrolyzed by alkaline phosphatase to yield phosphate and p-nitrophenol. (see next lecture)