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Rapid Mixing Techniques for Kinetics

المؤلف:  Wilson, K., Hofmann, A., Walker, J. M., & Clokie, S. (Eds.)

المصدر:  Wilson and Walkers Principles and Techniques of Biochemistry and Molecular Biology

الجزء والصفحة:  8th E , P497

2026-07-28

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 Rapid mixing techniques are required for processes with short timescale, such as fast kinetics, ligand binding and protein folding. These techniques involve turbulent mixing of two or more solutions in order to achieve homogeneous solutions in which processes can be monitored on a microsecond timescale.

In the continuous fl ow method, separate solutions of the enzyme and substrate are introduced from syringes, each of 10 cm 3 maximum volume, into a mixing chamber typically of 100 mm 3 capacity (Figure 13.22) . The mixture is then pumped through a narrow tube that is illuminated by a light source and monitored by a photomultiplier detector. The technique uses relatively small amounts of reactants and is limited only by the time required to mix the two reactants. The stopped- fl ow method is a variant of the continuous flow method. Shortly after the reactants emerge from the mixing chamber, the flow is stopped and the detector triggered to continuously monitor the change in an experimental parameter such as absorbance or fluorescence (Figure 1). Special flow cells are used together with a detector that allows readings to be taken 180° to the light source for absorbance, transmittance or circular dichroism measurements, or at 90° to the source for fluorescence, fluorescence anisotropy or light-scattering measurements. Yet another variation is the quenching method, where reactants from the mixing chamber are treated with a quenching agent from a third syringe. The quenching agent, such as trichloroacetic acid, stops the reaction, which is then monitored by an appropriate analytical method for the build-up of intermediates. By varying the time between mixing the reactants and adding the quenching reagent, the kinetics of this build-up can be studied. A disadvantage of this approach is that it uses larger amounts of reactants, since the kinetic data are acquired from a series of studies rather than by following one reaction for a period of time.

Fig1. Schematic comparison of the experimental set-up for continuous flow (a) and stopped-flow (b).