Stopped-flow experiments product formation

For a fast chemical reaction, such as the determination of calcium by o-cresolphthalein using colorimetric detection at 580 nm [253], shown in Fig. 2.32b, the product formation is so rapid that the response curve is identical with the dispersion curve obtained if product were injected as such. This is readily seen from the zero slope (dRIdt = 0) of the reaction rate curve A = B recorded with the element of fluid at the peak top (at CT , using delay time tdi). The same stopped-flow experiment repeated at the peak gradient (at Ca, using delay time r z) again has a zero slope... 

Nitrobenzofuroxan has been recently investigated in the isopro-poxide-isopropanol and cyanide-isopropanol systems.230 In both cases a reversible formation of a 7-adduct is followed by the stopped-flow method using similar spectral changes from substrate to adduct. The structure of the adduct is identified by analogy with the NMR and UV-visible properties of the product of the reaction as carried out with the MeO -MeOH system. There is no evidence that the formation of the 7-adduct is preceded by that of the 5-adduct under the conditions of the experiments. Any formation of the... 

A significant step to the combination of our knowledge about the static structure in liquids and their kinetic behavior has recently been made by application of an in-laboratory stopped flow EXAFS experiment [32]. This is an EXAFS spectrometer operated in the dispersive mode and a stopped-flow unit positioned along the x-ray path. Since results of very short time measurements can be accumulated in this way, with the appropriate selection of the system structural studies of reaction intermediates can be determined, which has not been possible before. Results are reported [33] on a partial structural change around the Cu(II) ion of a reaction intermediate at the formation of a Cu(II) porphyrin complex in the metal substitution reaction of the Hg(II) porhyrin complex with the Cu( o ion in an aqueous acetate buffer solution. The measurements showed that the Cu-N distance in the reaction intermediate are elongated by about 0.04A in comparison with the final product. 

The most serious limitation of stopped-flow methods is that one does not always have an optical signal for the reaction of interest and the optical signals cannot be interpreted rigorously if the extinction coefficients of intermediates or products are not known. For example, an enzyme intermediate may have an unknown extinction coefficient, and without an absolute measurement of concentration of the intermediate, one cannot obtain a unique solution to its rate of formation and decay (see below). For these reasons, a direct measurement of the conversion of substrate to product is required. Chemical-quench-flow methods allow such direct measurement of the chemistry of enzyme-catalyzed reactions and can be performed for nearly any reaction. One must recognize that these experiments are based on examining the enzyme as a stoichiometric reactant such that the concentration of enzyme required will depend upon the kinetics of the reaction and the sensitivity of the methods for detection of intermediates or products. Nonetheless, quench-flow experiments can be performed using as little as 20 fd of solution and a complete enzyme pathway can be solved using only... 

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