Rapid mixing techniques

The reaction of OH radicals with dimethyl sulfoxide in aqueous solution was studied already in 1964 by Norman and coworkers37 38. They used the system T1m-H202 to produce OH radicals and using ESR/rapid mixing techniques they were able to demonstrate elimination of a methyl radical during the OH induced oxidation. Further studies showed the formation of sulfmic radicals in this reaction either directly or by spin trapping experiments39-44. 

The permanganate oxidation of iodide has been the subject of a recent detailed study by a rapid mixing technique . At 35 °C and at an ionic strength of 0.9 M over the pH range 3-6 the rate expression is... 

Haight et al. ° have published a detailed account of the kinetics and stoichiometry of the oxidation of buffered bisulphite ion by chromic acid. The reaction is fast and its study requires a rapid mixing technique. The stoichiometry varies from a Cr(VI)/S(IV) molar ratio of 1 2 to 2 3 as the initial concentrations are changed in the range 0.12 < [Cr(VI)]/[S(IV)] < 1.4 and this was explained in terms of competition between two overall reactions... 

Free-radical intermediates in the oxidation of a series of quinol phosphates by permanganate at pH 11.7 have been characterised by an esr rapid-mixing technique . Clearly a one-equivalent oxidation step to give a semiquinone phosphate radical operates in this case. 

Raman scattering 192 Random mechanism 120 Rapid equilibrium mechanism 120 Rapid mixing techniques 133-136 Rapid quenching techniques 135-136... 

In sections A to C, we shall discuss four types of techniques. The first is rapid % mixing. This is extremely useful "since it is possible to mix two solutions in a fraction of a millisecond, and the majority of enzyme turnover numbers are less I than 1000 s 1. Rapid mixing techniques are now standard laboratory practice because of their ease and their wide range of application. 

The initial evidence for the formation of an acyl-enzyme ester intermediate came from studies of the kinetics with which chymotrypsin hydrolyzed analogs of its normal polypeptide substrates. The enzyme turned out to hydrolyze esters as well as peptides and simpler amides. Of particular interest was the reaction with the ester p-nitrophenyl acetate. This substrate is well suited for kinetic studies because one of the products of its hydrolysis, p-nitrophenol, has a yellow color in aqueous solution, whereas p-nitrophenyl acetate itself is colorless. The change in the absorption spectrum makes it easy to follow the progress of the reaction. When rapid-mixing techniques are used to add the substrate to the enzyme, an initial burst of p-nitrophenol is detected within the first few seconds, before the reaction settles down to a constant rate (fig. 8.8). The amount of p-nitrophe-... 

Such correlations are not always possible because the estimated rates at 25 °C may be too fast to measure with rapid-mixing techniques and because the rates of formation of more than one intermediate may be of the same order of magnitude at the higher temperature (12). 

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. 

Rapid chemical quench has been enormously powerful and helped to open the field of transient enzyme catalysis, as described above however, an alternative strategy is required for the detection and characterization of labile intermediates that avoids the need for chemical quenching. The coupling of rapid mixing techniques with online MS detection not only offers an option for the detection of labile intermediates but also has many general applications in the study of enzyme catalysis to more precisely define the key chemical events occurring at the active site. 

Secondly, rapid-mix techniques, in both the liquid and gas/liquid phases, indicated that oxygen had no radiosensitizing effect if the oxygen exposure occurred only a few milliseconds after the radiation [74]. This clearly indicated that short-lived species, probably free radicals, were involved. 

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