Martin Mangold discusses the hurdles and potential solutions for scientific experiments
Enzymes are proteins that can catalyse biochemical reactions. They 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 signals 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 data points over the complete chosen measurement time. This 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 a light signal. Here, the amounts of employed enzyme and substrate determine the time course of an enzyme kinetic. While an abundance of the 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. This 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 the determined Km. This is essential to ensure that the limiting factor is the activity of the enzyme itself and not the amount of available substrate, since its amount is enough for the enzyme to run at full capacity. For the final enzyme kinetic, ideally enzyme and substrate concentrations as well as measurement time are chosen, so that approximately 10% of the total substrate is converted during a test run.
Adjusting Microplate Reader Settings
Measuring devices such as microplate readers offer further possibilities to define the best measurement window for a kinetic measurement. First, depending on the assay, selecting appropriate excitation and/or emission wavelengths is crucial for the generation of significant readout signals. Selecting these wavelengths based on peaks in the excitation/emission spectra of the used substrate will lead to the best results, while deviating from them can result in drops in signal strength. To a certain degree, this effect can be compensated by increasing the flash number of the excitation light source in fluorescence-based measurements or increasing
the signal acquisition time window for assays which produce a long-lived emission signal, such as luminescence or time-resolved fluorescence measurements.
As most enzyme kinetics are based on the detection of fluorescent signals, one of the most important factors for setting the optimal assay window is an appropriate fluorescence gain value, which is the factor by which the incoming fluorescent signal is amplified. Typically, high gain values provide a large amplification and are hence suitable for dim signals. Very bright signals instead need a lower gain as less signal amplification is required (Fig. 2). Inappropriate fluorescence gain values negatively affect data quality, assay window and sensitivity. If bright samples are measured with a high fluorescence gain, this may result in the saturation of the detector and unusable data. On the contrary, if dim signals are detected with a low fluorescence gain, they may become indistinguishable from the background noise.
To avoid these issues and provide the best possible dynamic range between the highest and the lowest measurement values of your assay, fluorescence gain is typically adjusted on the sample with the expected highest signal output. However, kinetic assays pose a problem, as the light yield of the samples typically grows over time, increasing the chance of reaching saturation.
While all BMG Labtech microplate readers can automatically determine the fluorescence gain, the enhanced dynamic range (EDR) technology on the Clariostar Plus and the Vantastar was specifically designed to offer the largest possible dynamic range with no manual intervention. This makes gain adjustments superfluous as EDR ensures highly reliable results over a large dynamic range. Moreover, it simplifies the acquisition of kinetic experiments as it eliminates the risk of running into saturation.
Combining the above guidelines should give you a general idea for optimising kinetic enzyme studies.
Martin Mangold is with BMG Labtech