Rapid Kinetic Techniques

The elementary steps in complex biochemical reaction schemes (isomerization, dissociation, and association reactions) ultimately determine how fast any system can react in responding to incoming signals and in adapting to new conditions. Many of these steps have associated rate constants that result in subsecond responses to incoming signals or externally applied changes. This chapter is concerned with the techniques that have been developed to study such rapidly reacting systems in vitro and to determine the values of the rate constants for the individual steps. We focus principally on two classes of techniques: (1) flow techniques, in which two solutions are mixed within a few milliseconds and the ensuing reaction monitored over milliseconds to seconds, and (2) relaxation techniques, in which a small perturbation to an existing equilibrium is applied within a few microseconds and the response of the system is followed over microseconds to hundreds of milliseconds. These reactions are most conveniently monitored by recording the change in some optical signal, such as absorbance or fluorescence. We discuss the instrumentation that is (commercially) available to study fast reactions and describe a number of optical probes (chromophores) that can be used to monitor the changes. We discuss the experimental design appropriate for the different experimental techniques and reaction mechanisms, as well as the fundamental theoretical concepts behind the analysis of the data obtained.