Kinetics, stopped flow

The procedure which had originally been used by Lehn et al. involved slow addition (over a period of ca. 8 h) of ca. 0.1 M solutions of diamine and diacyl halide in benzene. Dye et al. found that the reactions could be conducted more rapidly as long as stirring was kept efficient. This observation suggested the use of a mixing chamber of the type normally used for stopped-flow kinetic studies. Utilizing this type of set-up, the latter authors were able to obtain a 70% yield for 1, slightly inferior to the yield reported by Lehn, but a similar yield of 3 which is better than that previously ob-tained. Note that the chemical features of this synthetic method are essentially identical to the approach shown in Eq. (8.1) and differ primarily in the mechanics. 

Figure 4-11. Schematic diagram of stopped flow kinetic system.

Abu-Soud, H., Mullins, L. S., Baldwin, T. O., and Raushel, F. M. (1992). Stopped-flow kinetic analysis of the bacterial luciferase reaction. Biochemistry 31 3807-3813. 

The stopped-flow and quenched-flow methods for fast reactions involve the fast flowing together of separate solutions of the reactants. This rapid mixing can be coupled to a rapid-response method for monitoring the progress of the reaction. With such methods one can determine rate constants up to about 5 X 102 s 1 (i.e., t n > 1 ms). The instrumentation for stopped-flow kinetics is readily available commercially. With special adaptations, one can gain another one or two orders of magnitude. 

Stopped-flow kinetics. If one uses an apparatus with a dead time of 2.3 ms, what fraction of a second-order reaction is missed if the initial concentrations are 2.0 X 10 3 M and 6.8 x 10 3 M, given a rate constant of 3.7 x 103Lmol 1 s-1 ... 

The mechanism of the first half-reaction has been studied by a combination of reductive titrations with CO and sodium dithionite and pre-steady-state kinetic studies by rapid freeze quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics 159). These combined studies have led to the following mechanism. The resting enzyme is assumed to have a metal-bound hydroxide nucleophile. Evidence for this species is based on the similarities between the pH dependence of the EPR spectrum of Cluster C and the for the for CO, deter-... 

Coles, B., Wilson, I. and Wardman, P. (1988). The spontaneous and enzymatic reaction of N-acetyl-/>-benzoquinone imine with glutathione, a stopped flow kinetic study. Arch. Biochem. Biophys. 264, 253-260. 

Kane DJ, Fendler K, Grell E et al (1997) Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase. Biochemistry 36 13406-13420... 

Stopped flow kinetic measurements indicate that when two aqueous solutions, one containing BaPDE and the other DNA, are mixed rapidly, a non-covalent site I-type complex is formed within 5 ms or less (11). 

Researchers studying the stepwise kinetics of nitrogenase electron transfer using stopped-flow kinetic techniques have presented other scenarios. One hypothesis presents kinetic evidence that dissociation of Fe-protein from MoFe-protein is not necessary for re-reduction of Fe-protein by flavodoxins.13 These authors state that the possibility of ADP-ATP exchange while Fe-protein and MoFe-protein are complexed with each other cannot be excluded and that dissociation of the complex during catalysis may not be obligatory when flavodoxin is the Fe-protein reductant. This leads to the hypothesis that MgATP binds to the preformed Fe-protein/... 

Many transition metal complexes have been considered as synzymes for superoxide anion dismutation and activity as SOD mimics. The stability and toxicity of any metal complex intended for pharmaceutical application is of paramount concern, and the complex must also be determined to be truly catalytic for superoxide ion dismutation. Because the catalytic activity of SOD1, for instance, is essentially diffusion-controlled with rates of 2 x 1 () M 1 s 1, fast analytic techniques must be used to directly measure the decay of superoxide anion in testing complexes as SOD mimics. One needs to distinguish between the uncatalyzed stoichiometric decay of the superoxide anion (second-order kinetic behavior) and true catalytic SOD dismutation (first-order behavior with [O ] [synzyme] and many turnovers of SOD mimic catalytic behavior). Indirect detection methods such as those in which a steady-state concentration of superoxide anion is generated from a xanthine/xanthine oxidase system will not measure catalytic synzyme behavior but instead will evaluate the potential SOD mimic as a stoichiometric superoxide scavenger. Two methodologies, stopped-flow kinetic analysis and pulse radiolysis, are fast methods that will measure SOD mimic catalytic behavior. These methods are briefly described in reference 11 and in Section 3.7.2 of Chapter 3. 

A detailed stopped-flow kinetics investigation (85) of this reaction (Por = TmTP and OEP) demonstrated that the mechanism occurs in two stages. The first was quite fast and was suppressed by the presence of excess CO and other coordinating ligands. Time resolved infrared... 

Fig. 5 Plausible mechanistic path for the oxidative addition of bromine to diphenylselenide, dicyanomethylidene telluropyran 20, and 2,6-diphenyltelluropyran-4-one (22) based on stopped-flow kinetics. An initial fast reaction followed second-order kinetics (first order in both bromine and substrate) while a second, slow reaction followed first-order kinetics. For diphenylselenide, a third very-slow reaction was observed.

M. Fabian and co-workers have studied the protein s role in internal electron transfer to the catalytic center of cytochrome c oxidase using stopped-flow kinetics. Mitochondrial cytochrome c oxidase, CcO, an enzyme that catalyzes the oxidation of ferrocytochrome c by dioxygen, is discussed more fully in Section 7.8. In the overall process, O2 is reduced to water, requiring the addition of four electrons and four protons to the enzyme s catalytic center. Electrons enter CcO from the cytosolic side, while protons enter from the matrix side of the inner mitochondrial membrane. This redox reaction. 

A study similar to that of Hersey and Robinson has been reported by Seiyama and coworkers.From a stopped-flow, kinetic study of the interaction of various azo dyes and some azo dye-metal complexes with alpha cyclodextrin, they observed two kinetic processes. The dependence of the observed rate-constants for these two processes on the alpha cyclodextrin concentration was found to be explainable in terms of a mechanism identical to that proposed earlier by Hersey and Robinson. In the case of the guests used by Seiyama and coworkers, however, values for the rate constants of the binding step could be determined from the concentration dependence of kobs for the faster process thus,... 

The kinetics of myoglobin oxidation and reduction have been studied by a variety of experimental techniques that include stopped-flow kinetics, pulse radiolysis, and flash photolysis. In considering this work, attention is directed first at studies of the wild-type protein and then at experiments involving variants of Mb. 

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