Substrate Depletion or Product Accumulation

When a substrate can be directly measured, the reason for using an enzymatic assay for its quantitation may not be immediately apparent. However, complex biological media matrices may contain a variety of species that interfere with the direct measurement of analyte concentration. For example, if the analyte absorbs in the visible (vis) or ultraviolet (UV) region, direct quantitation may result in erroneously high values if interfering species absorb at the measurement wavelength. An enzymatic method, on the other hand, can monitor the absorbance decrease that occurs as a result of the selective consumption of analyte by the enzyme, thus avoiding the spectral interference. 

An assay for uric acid involves the enzyme urate oxidase, which catalyzes the following reaction  

While allantoin is difficult to measure spectrophotometrically, uric acid possesses a strong UV absorption with a maximum at 293 nm (molar absorptivity 1.22 x 104A/-1cm-1).1 Uric acid quantitation thus involves monitoring the decrease in A293. 

Coenzyme A (CoA) may be quantitated using the enzyme phosphotransacety-lase, which catalyzes the acetylation of CoA by acetylphosphate  

In this case, CoA—S—COCH3 accumulation is measured, since it possesses an absorption maximum at 232 nm (molar absorptivity 4.5 x 103 M x cirT1).2 The parameter, 42 2 will therefore increase as the reaction proceeds. 

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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. 

Absorbance. The detection of substrate depletion or product accumulation through the measurement of visible or UV fight absorbance is based on the Beer-Lambert law, which directly relates absorbance at a given wavelength to concentration ... 

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