methods is to insert a fluorine atom into the molecule. From an NMR point of view
this is an excellent label, since it is a spin-half nuclide that is readily studied by NMR.
The chemical shift change of the fluorine nucleus is large, making it very sensitive
to its local environment in the protein. Moreover, its size is very similar to that of
a proton, so that it is unlikely to modify the enzyme’s structure. Since fluorine is
very rare in biological systems, the NMR signal from the label can be interpreted
unambiguously. By studying the relaxation times associated with the fluorine nucleus
it is possible to detect restricted motion of the enzyme in a cell due to protein–protein
aggregation.15.3.5 Analytical methods for substrate assays
Enzyme-based assays are very convenient methods for the estimation of the amount
of substrate present in a biological sample. The principle of using excess enzyme (i.e.
the substrate concentration should be less than theKm) and relating the substrate
concentration in the test solution to the observed initial rate can be used. It is essential
that the reaction goes to completion in a relatively short time. If the reaction is freely
reversible, then it is necessary to change the experimental conditions, such as pH or
by chemically trapping the product, so that the reaction does approach completion.
Coupled reactions are commonly used in substrate assays and they have the attraction
that they help in the displacement of reversible reactions. The sensitivity of this initial
rate method to substrate assay depends upon the value of the molar extinction
coefficient for the analyte being assayed and also on theKmfor the substrate. In
practice these two factors place a constraint on the level of substrate that can be
assayed. Several approaches are available to overcome this problem. Theend-point
techniqueavoids the measurement of initial rate by converting all the substrate to
product and then computing the amount present by correlating it with the total
change in parameter such as absorbance or fluorescence. The sensitivity of an assay
can also be significantly increased by the technique ofenzymic cycling. In this
method the substrate is regenerated by means of a coupled reaction and the total
change in absorbance, etc. in a given time measured. Precalibration using a range of
substrate concentrations with all the other reactants in excess allows the substrate
concentration in a test solution to be computed. This method has a 10^4 -to10^5 -fold
increase in sensitivity relative to the end-point technique.
Enzyme-based assays are commonly used in clinical biochemistry to measure
substrates in biological samples. For example, the three most common assays for
serum glucose are those based on the use of hexokinase, glucose oxidase and glucose
dehydrogenase. The first two are based on the coupled reaction technique:- Hexokinase method: This couples the reaction to that of glucose-6-phosphate
dehydrogenase and measures the absorbance at 340 nm due to NADH:
D-glucoseþATPÐD-glucose-6-phosphateþADP
D-glucose-6-phosphateþNADþÐD-glucono-1,5-lactone-6-phosphateþNADHþHþ610 Enzymes