uring an enzymatic reaction, a product is produced, a substrate is used, and the catalyzing enzyme remains unconsumed. Therefore, to analyze the kinetics of an enzyme, a measure of the rate of change in concentration of the substrate being used up and/or the product being formed is typically determined. Conventional parameters such as the Michaelis-Menten constant (KM) and maximum velocity (Vmax) require the concentration of an enzyme to be static while various concentrations of substrate are examined.1 Spectrophotometric assessment of the change in concentration at the start of the enzyme–substrate reaction is preferred for an accurate representation of the kinetics.1
Procedural Tip: Get it Right from the Start
The beginning of an enzyme–substrate reaction is critical for the estimation of KM
due to the quick and linear increase in concentration of the product known to occur.1
Of course, as the substrate is consumed over the course of the reaction, the rate of product accumulated decreases. Accurate steady-state enzyme kinetic measurements depend on this initial linear rate (a.k.a. presteady state), although nonlinear relationships can also be used by taking change-of- rate measurements at the reaction’s completion.2
There are, however, two mutually-dependent certainties standing in your way: 1) enzymes work at a speed where the initial rate may only be a few seconds, making 2) the quick speed required for mixing enzyme and substrate in different cuvettes at once very difficult when done manually. Real-time measurement of the initial stage of an enzyme–substrate reaction is essential for reproducible enzyme kinetics data. Automated direct-flow injection systems couple with various detection and analytics platforms to offer the speed, precision, and flexibility required to carry out the reaction.
Going with the Flow: Automated Syringe Pump Solutions
There are three commonly used strategies of flow that injection devices use, continuous, stopped, or quench flow.1 Their primary difference addresses the desired response after the substrate and enzyme are mixed. Continues refers to a flow that is not interrupted; a stopped-flow will hold the mixture in a spectrophotometric-specific chamber, whereas a quenched flow requires a psychical or chemical stop to the reaction at the exit of the device for additional analyses.1-3 Their advantages and disadvantages have been summarized at length.1
Traditionally, researchers have prepared mixtures of enzyme and varying concentrations of substrate at their bench, followed by immediate spectrophotometric recording. The accuracy of the manual process is limited by inconsistencies in mixing time, transport to and from cuvette(s), and into and out of the spectrophotometer, thereby losing valuable analysis time at the critical initial stage of the reaction.
Syringe-operated flow apparatuses are an automated alternative that quickly and precisely introduce the sample to the detection machine, thus improving your analysis time. These systems also offer high-throughput features. The computer-programmed syringe-pump platform allows for the examination of enzyme kinetics using multiple substrate concentrations, saving you not only time but also providing you with superior predictive and conclusive data about enzyme activity and characteristics. Commercially available models can be integrated into detection equipment (like a spectrophotometer) accompanied with software, or into analytical diagnostic, microfluidic instruments making them ideal for a multitude of applications.
1. S.K. Hartwell and K. Grudpan, “Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies,” J of Anal Methods Chem 2012:1-10, 2012.
2. C.T. Houston, et al., “Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry,” Anal Chem 15:3311-3319, 2000.
3. C. Balny, “Enzyme Kinetics: Stopped-Flow Under Extreme Conditions,” In: Taniguchi Y., Stanley H.E., Ludwig H. (eds) Biological Systems Under Extreme Conditions. Biological and Medical Physics Series. Springer, Berlin, Heidelberg, pp.167-185, 2002.
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