Enzyme Technology
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 Km,
equation 1.8 simplifies to give
v =
(Vmax/Km)[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 Km of the enzyme for galactose. In
contrast, the NAD+ concentration is kept within a concentration range
well above the Km 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.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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