Stopped-Flow Kinetic Methods

In order to use the stopped-flow technique, the reaction under study must have a convenient absorbance or fluorescence that can be measured spectrophotometri-cally. Another method, called rapid quench or quench-flow, operates for enzymatic systems having no component (reactant or product) that can be spectrally monitored in real time. The quench-flow is a very finely tuned, computer-controlled machine that is designed to mix enzyme and reactants very rapidly to start the enzymatic reaction, and then quench it after a defined time. The time course of the reaction can then be analyzed by electrophoretic methods. The reaction time currently ranges from about 5 ms to several seconds. 

TN was increased by the presence of the general acid. These observations suggested that H2O serves to donate the protons required to form product H2O2. Values of Km and TN for the zinc-deficient enzyme were found to be approximately a factor of two less than those obtained for the holoenzyme under identical experimental conditions, whereas TN/Xm was largely unchanged. The authors concluded that the imidazolate bridge is thus not essential for catalytically competent extraction of a proton from the solvent by CuZnSOD. 

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. 

---

Sequential Discontinuous Stopped-flow kinetic methods... 

No burst. Examination of the cleavage of the chromogenic amide substrate, A, by chymotrypsin with the use of stopped-flow kinetic methods reveals no burst. Why ... 

Intermediates in the reaction of the dioxygenase ES complexes with O2 can be observed using stopped-flow kinetic methods. Two transient species are observed and both exhibit tyrosinate-to-iron(III) charge transfer bands, though the band shifts in energy... 

A more thorough evaluation of the dynamics of tertiary aminium radical a-CH deprotonation reactions has come from relatively recent studies employing stopped-flow, time-resolved laser flash photolysis, and electrochemical techniques. In one effort, Dinnocenzo and Banach examined acid-base reactions of the stable aminium radical 3, prepared by SET oxidation of N,N-dianisyl-N-methylamine (Scheme 5), Using stopped-flow kinetic methods, these workers showed that the bimolecular rate constants for a-deprotonation of 3 by four quinuclidine bases 4 in MeCN are (1) in the range of 1 x 10 to 6 x 10 M ... 

---