Direct and Coupled Measurements

Some enzymatic reactions can be followed directly, either by substrate depletion or product accumulation measurements, with adequate precision for direct enzymatic assays. However, many enzymes catalyze reactions involving species that are not themselves readily measured. In these situations, products are converted to species that are measurable, in a coupled, or indicator reaction. The indicator reaction may be chemical or enzymatic, and quantitatively converts the product of the primary reaction into a readily measurable species. The main requirement for the indicator reaction, whether it is chemical or enzymatic in nature, is that the conversion of the primary product into the measured product must be rapid and quantitative. 

An assay for adenosine involves the primary enzyme adenosine deaminase, and a chemical indicator reaction that consumes ammonia by reaction with the ninhy-drin reagent (Eq. 3.4)  

The product of this reaction absorbs visible light, with a maximum at 546 nm. The ninhydrin indicator reaction3 may be employed with virtually any primary reaction that produces NH3. 

The most commonly used indicator enzymes are dehydrogenases and peroxidases. The reactions catalyzed are shown in Eq. 3.5. 

Reduced substrate + NAD(P)+ dehydr°senase Oxidized substrate + NAD(P)H + H+ 

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