Stopped-flow spectroscopy

With the advent of powerful, fast computers it has now become possible to process very large data sets quickly and this has paved the way for the application of rapid scan devices (usually, but not exclusively, diode arrays), which allow complete spectra to be collected at very short time intervals during a reaction. In these circumstances there is a danger of being swamped by data. 

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Table 2 Arrhenius and Eyring activation parameters for the second, Slow reaction observed by stopped-flow spectroscopy in the oxidative addition of halogens to diorgano tellurides 17, 20, and 23-25...

The oxidative addition of iodine to diorganotellurides has also been examined by stopped-flow spectroscopy. The initial fast reaction of iodine with diphenyltellur-ide (23), di-4-methoxyphenyltelluride (24), A,V-dimethyl-2-(aminomethyl)phenyl-telluride (17), and 2,6-di-tert-butyltelluropyran-4-one (25) displays inverted Arrhenius behavior (negative values of E ), which is consistent with a preequilibrium involving higher-order iodine species as shown in equation (6). The I4 species is the actual oxidant for diorganotellurides as shown in equation (7). Thus, the initial reaction is formation of the r i-association complex of I4 with the... 

The catalytic cycle for the thiolperoxidase and haloperoxidase-like activity of diorganoselenides and tellurides is summarized in Fig. 25. Stopped-flow spectroscopy has been used to elicit mechanistic details of the cycle. " " Following oxidation to the selenoxide or telluroxide, the catalytic cycle for thiolperoxidase-like activity is shown in Fig. 21. The details of the haloperoxidase-like cycle are not as well defined. Using dihydroxytellurane 52 as a substrate, the addition of 0.5 M sodium iodide in pH 6.8 buffer gave a fast reaction with a second-... 

Of course, high reactivity of ion-radicals dictates some complexity in the registration methods, such as stop-flow spectroscopy or isolation of ion-radicals by capillary electrophoresis. Whereas stop-flow spectroscopy is used more or less frequently, capillary electrophoresis has been involved only in recent times. Choosing the appropriate capillary length, a researcher can isolate and register UV spectra of relatively less-stable ion-radicals. When the interaction of the corresponding... 

The detailed mechanism of P aeruginosa CCP has been studied by a combination of stopped-flow spectroscopy (64, 65, 84, 85) and paramagnetic spectroscopies (51, 74). These data have been combined by Foote and colleagues (62) to yield a quantitative scheme that describes the activation process and reaction cycle. A version of this scheme, which involves four spectroscopically distinct intermediates, is shown in Fig. 10. In this scheme the resting oxidized enzyme (structure in Section III,B) reacts with 1 equiv of an electron donor (Cu(I) azurin) to yield the active mixed-valence (half-reduced) state. The active MV form reacts productively with substrate, hydrogen peroxide, to yield compound I. Compound I reacts sequentially with two further equivalents of Cu(I) azurin to complete the reduction of peroxide (compound II) before returning the enzyme to the MV state. A further state, compound 0, that has not been shown experimentally but would precede compound I formation is proposed in order to facilitate comparison with other peroxidases. 

Ritchie was the first to directly measure the absolute reactivity of cations toward solvent and added nucleophiles. The cations were highly stabilized examples, triarylmethyl cations bearing stabilizing substituents such as 30 and 31, xanthylium ions (e.g., 32) and tropylium ions (e.g., 33). The feature (and requirement) of these cations was that they had a lifetime in water such that kinetics could be followed by conventional or stopped-flow spectroscopy whereby one solution containing the pre-formed cation was added to a second solution. The time required to mix these solutions was the important factor and limited measurements to cations with lifetimes longer than several milliseconds. The lifetimes in water for 30-33 are provided below. Lifetime is defined as the reciprocal of the first-order rate constant for the decay of the cation in solvent. 

Kissner R, Beckman JS, Koppenol WH (1999) Peroxynitrite studied by stopped-flow spectroscopy. Methods Enzymol 301 342-352... 

The electron transfer reaction from copper to heme within the ternary protein complex was also studied in solution by stopped-flow spectroscopy. Analysis by Marcus theory of the temperature dependence of the limiting first-order rate constant for the redox reaction (Davidson and Jones, 1996) yielded values for the of 1.1 eV and H b of 0.3 cm , and predicted an electron transfer distance between redox centers which was consistent with the distance seen in the crystal structure. Thus, the electron transfer event is rate-limiting for this redox reaction. Experiments are in progress to determine the validity of the predicted pathways for electron transfer shown in Figure 7. 

The u.v.-visible spectrum of the coenzyme changes significantly upon homolysis of the CooC bond AdoCbl is pink, a characteristic of the 6-coordinate Co(III) atom, whereas Cbl(II) is yellow-brown due to the presence of the 5-coordinate Co(II) species. This property provides a convenient and sensitive spectroscopic probe with which to monitor the enzyme-catalyzed homolysis of AdoCbl, the key step which results in the generation of free radicals. Thus, for several enzymes the rate of AdoCbl homolysis has been measured by stopped flow spectroscopy. 

As stated previously, early work into the direct observation of unstable Cuf-02 species met with little success. These species were often postulated based on both chemical logic, as well as limited kinetic information, but direct evidence pointing to the presence of such complexes was lacking. This situation changed in the mid-1980s in large part due to the utilization of low-temperature (-40 to -105 °C) stopped-flow spectroscopy where direct observation of spectroscopically identifiable copper-dioxygen intermediates was possible. This has allowed for the direct observation of a number of elementary steps in the formation and subsequent reactions of these complexes. 

Stopped-flow spectroscopy was used to relate the rate constants, activation parameters, and mechanisms of the oxidation of CIO2 and Br02 by O3. Ozone reactions with XO2 (X = Cl or Br) are studied under pseudo-first-order conditions with excess X02. The kinetic traces were obtained observing the loss of O3 at 260 nm and fitted to an exponential equation to give the pseudo-first-order rate constants. The 03/X02 reactions are first order in [O3] and... 

Only recently, studies of the non-stationary polymerization of styrene and its derivatives using the flow and stopped-flow spectroscopy have allows the determination of the rate constants in a more reliable way ... 

In addition to standard methods of monitoring the reaction progress by UV and visible spectroscopies, other detection methods also can be used. For example, electron-transfer reactions between monomeric and dimeric metal carbonyl complexes in Eq. 11 have been studied by infrared stopped-flow spectroscopy utilizing a tunable CO laser as a source of infrared radiation and a HgCdGe detector [12]. 

Silverman s studies on mechanism based MAO inactivation have provided overwhelming support for the role of electron transfer in the MAO catalyzed dealkylation of amines. It must be mentioned however that spectroscopic attempts for detecting the radical ion intermediates have hitherto been unsuccessful. Yasanobu and coworkers could not find EPR spectral evidence for radical intermediates in MAO-catalyzed oxidation of benzylamine [205]. Miller et al. failed to observe the flavin semiquinone or an amine-flavin adduct in rapid-scan-stopped flow spectroscopy [206]. The only time-dependent absorption change observed in this study was the bleaching of the oxidized flavin. Furthermore, no influence of a magnetic field up to 6500 G was observed on the rate of MAO B reduction. The reaction rates of systems with kinetically significant radical pair intermediates are known to be altered... 

Direct assays for SOD have relied on pulse radiolysis (K12.M15), stopped-flow spectroscopy (M4,M17), rapid-flow electron paramagnetic resonance (EPR) (Bl,05, SI), polarographic techniques (R4), and nuclear magnetic resonance (NMR) spectroscopy (R3). 

Dunn, Michael, Rapid-Scanning Stopped-Flow Spectroscopy. 37 191... 

Rapid-Scanning Stopped-Flow Spectroscopy Peter S. Brzovic and... 

A new method for the study of nonstationary polymerization is the flow and stopped-flow spectroscopy developed by Sawamoto and Higashimura (161. 162). Although these methods offer the only available data about polymerization kinetics through known species, their preparative applications are very limited. A number of useful discoveries are coming from Kennedy s laboratory (163-175). Part of these discoveries will be presented later. Kennedy s research philosophy consists in understanding the mechanisms of polymerization of conventional monomers, and its use in the design of new polymeric materials (163) has proven very productive. 

Significant Applications Use of Rapid-Scanning Stopped-Flow Spectroscopy to Investigate Enzyme Structure-Function Relationships... 

The kinetics of the oxidations of trans-[Tc(dppe)X2] (X = Cl , Br ) by Co(III) amine complexes have been investigated in pure and mixed DMF and CH3CN solvent systems, with an observed increase in the rate constant with DMF concentrations. The rate constants for the reductions of three metal carbonyl dimers [M2,M = Mn(CO)5, CpMo(CO)3, and Co(CO)4] by [Re(CO)5r in THF (measured using infrared stopped-flow spectroscopy) do not parallel the reduction... 

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