Rapid Sample-mixing Technique

Enzyme linked electrochemical techniques can be carried out in two basic manners. In the first approach the enzyme is immobilized at the electrode. A second approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of product to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system which is amenable to in vivo analysis. 

The use of more dilute lipid dispersions, unilamellar vesicles and, ultimately, cell suspensions is another direction worth pursuing. Such possibilities become more realistic as incident photon flux increases and more sensitive X-ray detectors become available. Since conventional rapid-mixing techniques can be used with more dilute suspensions, an enormous benefit will accrue in studies where the kinetic event is triggered by a jump in sample composition. 

The parameter y ax can be converted to a turnover number or kcat by taking account of the molar concentration of enzyme present in the assay mixture. The values of kcat of enzymes are generally in the range of 10 -10 12] means that the catalytic events on the enzyme occur in the time range of milliseconds or less. In order to characterize such steps, it is necessary to employ rapid reaction techniques such as stopped-flow, which has a dead time of approximately 1 ms. Continuous-flow mixing techniques can have shorter dead times but make much greater demands in terms of quantities of sample required. The value of kcJK provides not only a quantitative measure of the specificity of an enzyme for a given substrate but it can also be used as a measure of catalytic efficiency... 

In conclusion, rapid-mixing/rapid-freezing EPR is a wonderful technique to obtain unique molecular structural information on biochemical reaction intermediates with high time resolution, but it is also experimentally sufficiently involved that one should either build up a dedicated lab with dedicated operators or turn to one of the existing groups that have the equipment and, especially, the developed skills to do these experiments. Be prepared to provide at least an order of magnitude more sample than required for a static EPR experiment. 

B. Chance (1964) in Rapid Mixing and Sampling Techniques in Biochemistry (B. Chance, R. Eisenhardt, Q. H. Gibson K. Lonberg-Holm, eds) p. 125, Academic Press, New York. 

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