The use of enzymes in analysis

Enzymes make excellent analytical reagents due to their specificity, selectivity and efficiency. They are often used to determine the concentration of their substrates (as analytes) by means of the resultant initial reaction rates. If the reaction conditions and enzyme concentrations are kept constant, these rates of reaction (v) are proportional to the substrate concentrations ([S]) at low substrate concentrations. When [S] < 0.1 K_m, equation 1.8 simplifies to give

v = (V_max/K_m)[S]            (6.1)

The rates of reaction are commonly determined from the difference in optical absorbance between the reactants and products. An example of this is the b-D-galactose dehydrogenase (EC 1.1.1.48) assay for galactose which involves the oxidation of galactose by the redox coenzyme, nicotine-adenine dinucleotide (NAD⁺).

b-D-galactose + NAD⁺ D-galactono-1,4-lactone + NADH + H⁺            [6.1]

A 0.1 mM solution of NADH has an absorbance at 340nm, in a 1 cm path-length cuvette, of 0.622, whereas the NAD⁺ from which it is derived has effectively zero absorbance at this wavelength. The conversion (NAD⁺ NADH) is, therefore, accompanied by a large increase in absorption of light at this wavelength. For the reaction to be linear with respect to the galactose concentration, the galactose is kept within a concentration range well below the K_m of the enzyme for galactose. In contrast, the NAD⁺ concentration is kept within a concentration range well above the K_m of the enzyme for NAD⁺, in order to avoid limiting the reaction rate. Such assays are commonly used in analytical laboratories and are, indeed, excellent where a wide variety of analyses need to be undertaken on a relatively small number of samples. The drawbacks to this type of analysis become apparent when a large number of repetitive assays need to be performed. Then, they are seen to be costly in terms of expensive enzyme and coenzyme usage, time consuming, labour intensive and in need of skilled and reproducible operation within properly equipped analytical laboratories. For routine or on-site operation, these disadvantages must be overcome. This is being achieved by the production of biosensors which exploit biological systems in association with advances in micro-electronic technology.