Stopped flow kinetic measurements

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). 

The kinetics of the formation of carbonato complexes by CO2 uptake reaction has been extensively investigated (3). The two methods of study, the equilibrium method and acidification method , genuinely devised by Harris and co-workers were adapted to the stopped-flow kinetics measurement (63). The following general reaction scheme was proposed by them for the formation of monodentate carbonato/bicarbonato complexes by the C02 uptake reaction of M-OH(K-1)+ species (Scheme 2). 

High-pressure stopped-flow kinetic measurements were performed up to 200 MPa. The observed rate constants, ki,obs and k2,obs as a function of pressure and a-CD concentration, were fitted simultaneously according to the reaction scheme shown in Fig. 11.8 leading to the microscopic rate constants kij, k2,f, h,r and fe2,r-The results are summarized in the form of volume profiles in Fig. 11.10. 

The half-life for nonenzymic interconversion of the anomers is about 0.4 sec. Stopped-flow kinetic measurements, using the isomerase from yeast, indicate that both the a- and j8-anomers of glucose 6-phosphate are used directly by the enzyme to give the a- and )8-anomers of fructose 6-phosphate, although the a-anomer is consumed at least 20-fold faster than the )3-anomer. In addition, the enzyme is capable of catalyzing the interconversion (anomerization) of the a-and jS-anomers. With the a-anomer of glucose 6-phosphate, the anomerization reaction is approximately twice as fast as the isomerization reaction. 

We have gathered compelling evidence supporting the mechanism of hydroxylation to be one where the reaction occurs via an electrophilic attack on the juxtaposed aryl group. Stopped flow kinetic measurements of the oxidation of substituted [Cu2(R-XYL-H))] show that k ydroxyi (Fig. 25) increases as R becomes more electron donating i.e., stabilizing... 

Stopped-flow kinetic measurements under conditions of enzyme excess have shown that a rapid increase in mansyl fluorescence on mixing a substrate with pepsin is followed by a first-order decrease in fluorescence when the affinity of the acidic cleavage product for the active site is much less than that of the substrate. Such kinetic measurements have given estimates of Kg, the dissociation constant of the rate-limiting Michaelis complex, and in all cases = Kjj, the Michaelis constant estimated under conditions of substrate excess by determination of the rate of formation of the amine product (24). This result strengthens the validity of earlier conclusions (3,13) that the rate-limiting step in the action of pepsin on peptide substrates under conditions of substrate excess is the decomposition of the first detectable enzyme-substrate complex, and that no kinetically significant intermediate accumulates in the process. 

A number of recent papers have also focussed on the identification of paramagnetic transient reactive intermediates in bio-inspired non-heme iron catalysed oxidations. Makhlynets and Rybak-Akimova investigated the mechanism of the substrate oxidation, using H2O2 as oxidant, in an iron aminopyridine complex for aromatic hydro qrlation (Scheme 1). EPR with stopped-flow kinetic measurements, was used to identify the formation of the key Fe" (OOH) intermediate, with rhombic g... 

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 comprehensive series of oxidation-reduction potential measurements have shown the FAD moiety to have the following one-electron couples PFl/PFIH = = —290 mV and PFIH 7PFIH2 = —365 mV while the FMN moiety exhibits the following PFl/PFl- = -110 mV and PFIH /PFIH = -270 mV. The FMN and FAD smiquinones were found to both be the neutral form as judged from absorption and ESR spectral data. The overlap of oxidized/semiquinone potential of the FAD moiety withkhe semiquinone/hydroquinone couple of the FMN moiety demonstrates the thermodynamic facilitation of flavin-flavin electron transfer via a one-electron mechanism. Stopped-flow kinetic data are also consistent with this view in... 

The energetics of protein association can be studied by a variety of experimental techniques,17 each of which permits measurements of equilibrium or kinetic values in a certain range. Widely used techniques include isothermal titration calorimetry, surface plasmon resonance measurement, stopped flow kinetics, optical spectroscopy, MS, and analytical ultracentrifugation. The techniques differ in their requirements (e.g., amount of protein, labeling with fluorophores, attachment to sensor surfaces, and the environment provided by the experimental set up) and therefore in their applicability to individual cases. Different techniques can also give quite different values for what might be expected to be the same quantity. For example, association rates measured by surface plasmon resonance, with one protein immobilized on a surface, are usually different from those measured for the two proteins in solution and under otherwise similar conditions. 

Figure 15-6 Stopped flow kinetic traces of Fe(II) oxidation by some ferritins and their variants. Assays involved the addition of an equal volume of ammonium iron (II) sulfate in SOpM H2SO4 to an aerobic solution of the iron-free protein (final cone. 1 pM except for EcBfr which was 0.2S pM) in 0.2 Mes buffer, pH 6.5, to give 48 Fe(Il) atoms per molecule. HuHF and HuHF E61 A absorbance at 370 nm not measured, EcBfr and EcBfr-M52H absorbance at 550 nm not above baseline. (A) HuHF (B) HuHF-E61A (C) EcBFR-M52H (D) EcFTNa (E) EcFTNa-E49A (F) EcFTNa-E130A.

---