MICROPLATE READERS
ENZYME KINETICS CONSIDERATIONS
Martin Mangold discusses the hurdles and potential solutions for scientific experiments
BMG Labtech’s latest microplate readers are ideally suited for enzyme kinetics
E
nzymes are proteins that can catalyse biochemical reactions. Tey are particularly important due to their role in the synthesis, modification and degradation of organic matter. Since enzymatic reactions are not static, enzyme activity is generally monitored over time in kinetic measurements to record the changing assay readout. However, recording these changes in assay signal can provide certain challenges and hurdles. Here, we discuss how to define the perfect measurement window for enzyme kinetics and which solutions modern microplate readers offer for this purpose.
DETERMINATION OF KINETIC PARAMETERS Ideally, a measurement device should detect and display an assay signal for all
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data points over the complete chosen measurement time. Tis can be achieved by two means, either by adjusting the settings of the measurement device or by adjusting the assay itself.
Assay parameters can be adjusted by changing the concentrations of the assay reagents. Generally, the components for enzyme activity assays comprise the enzyme of interest, either in purified form or as part of a biological sample solution, an assay buffer and an enzyme substrate. In typical colorimetric and fluorescent assays, cleavage of the substrate by the enzyme of interest leads to the generation of light signal. Here, the amounts of employed enzyme and substrate determine the time course of an enzyme kinetic. While an abundance
of enzyme can lead to a faster cleavage of substrate, inappropriate substrate concentrations can limit the assay signal output. An important measure for choosing appropriate substrate concentrations for kinetic measurements
is the determination of a substrate´s Km value. Tis so-called Michaelis constant reflects the affinity of an enzyme for the substrate and represents the substrate concentration at which the half-maximal velocity of the enzymatic reaction is reached. It can be determined by plotting the slope of the enzymatic reaction against increasing substrate concentrations (Fig. 1). In practice, when running an enzyme activity assay, it is common to use an about 10- to 20-fold higher concentration of substrate than
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