As is apparent from previous chapters (Chapters 6, 8 and 9), understanding the kinetics of chemical and biological processes is extremely important. Questions we often consider, explicitly or implicitly, include: Has something happened ‘instantaneously’ or will it take 20 years? Does changing the conditions or available reagents affect either the end product or the rate of a process? What intermediates are produced during a reaction? Can we characterize any intermediates? Do we need to remove them to prevent side reactions? If some or all of the reactants or products are chiral, then circular dichroism (CD) detection may be the ideal tool for following the kinetics of a reaction, and if the half-life of the reaction is of the order of milliseconds to seconds or even minutes then stopped-flow mixing of the reagents will almost certainly be the appropriate choice of sample handling method. For reactions with half-lives of a few minutes to tens of minutes the reagents can be mixed by hand in a normal cuvette and the signal monitored at an appropriate wavelength. CD is not well suited to kinetics on timescales of hours due to the baseline drift that does occur (see Chapter 4, Section 2.5). Some CD spectropolarimeters have the useful facility of being able to perform a wavelength scan at pre-set intervals as well as monitoring continuously (except during the wavelength scan) at a chosen wavelength, thus facilitating the characterization of any intermediates. In this chapter we shall highlight some of the considerations of the stopped-flow technique that are particularly relevant to CD experiments. Particular problems may be encountered when performing CD (as opposed to other detection methods) stopped-flow experiments. The measured signals are very small (typical CD intensities are 0.1% or less of the absorbance signal), and the noise level observed is particularly sensitive to any inhomogeneities or turbulence in the samples. Also, as one of the main applications of stopped-flow CD is in the study of protein folding and unfolding, samples are often very viscous and/or corrosive, have significant absorbances due to buffers etc., and the experiments often require wide and variable mixing ratios.
Observation and time resolution of chiral thiamin diphosphate‐bound intermediates in the catalytic cycle of pyruvate decarboxylase and benzoylformate decarboxylase by stopped‐flow circular dichroism.
