EPR monitored kinetics

In principle, EPR spectrometry is well suited as a method to monitor kinetic events however, in practice, the time required to tune the spectrometer, and its intrinsically low sensitivity compared to fluorescence or light-absorption spectrometry, affect its competitiveness. Relatively slow reactions on the timescale of minutes, such as the decomposition of the DMPO-superoxide adduct and the subsequent formation of the hydroxyl radical adduct (cf. Pou et al. 1989) are readily followed, either as the first-order disappearance of the DMPO/ OOH signal 

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. 

Finally, as a poor man s alternative, consider the possibility to slow down the reaction kinetics by running a reaction at 0°C temperatures (especially by employing enzymes from hyperthermophilic species) so that the mixing may be done in seconds (by hand ), and let us then hope that the kinetic mechanism under these nonphysiological conditions still bears relevance to the natural biology. 

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Characterize EPR-monitored kinetics down to the 0.5 s dead time in your own lab. 

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