title: Rapid mixing kinetic techniques.
abstract: Almost all of the elementary steps in a biochemical reaction scheme are either unimolecular or bimolecular processes that frequently occur on sub-second, often sub-millisecond, time scales. The traditional approach in kinetic studies is to mix two or more reagents and monitor the changes in concentrations with time. Conventional spectrophotometers cannot generally be used to study reactions that are complete within less than about 20 s, as it takes that amount of time to manually mix the reagents and activate the instrument. Rapid mixing techniques, which generally achieve mixing in less than 2 ms, overcome this limitation. This chapter is concerned with the use of these techniques in the study of reactions which reach equilibrium; the application of these methods to the study of enzyme kinetics is described in several excellent texts (Cornish-Bowden, Fundamentals of enzyme kinetics. Portland Press, 1995; Gutfreund, Kinetics for the life sciences. Receptors, transmitters and catalysis. Cambridge University Press, 1995).There are various ways to monitor changes in concentration of reactants, intermediates and products after mixing, but the most common way is to use changes in optical signals (absorbance or fluorescence) which often accompany reactions. Although absorbance can sometimes be used, fluorescence is often preferred because of its greater sensitivity, particularly in monitoring conformational changes. Such methods are continuous with good time resolution but they seldom permit the direct determination of the concentrations of individual species. Alternatively, samples may be taken from the reaction volume, mixed with a chemical quenching agent to stop the reaction, and their contents assessed by techniques such as HPLC. These methods can directly determine the concentrations of different species, but are discontinuous and have a limited time resolution.
authors.affiliation: Division of Physical Biochemistry, MRC National Institute for Medical Research, London, UK.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Martin
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Schilstra
mesh_terms: D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008968:Molecular Conformation; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D011506:Proteins