Stopped pulse radiolysis

Cabelli, D.E. and Bielski, B. (1983). Kinetics and mechanism for the oxidation of ascorbic acid (ascorbate by HO2/O2 radicals. A pulse radiolysis and stopped-flow photolysis study. J. Phys. Chem. 87, 1809. 

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. 

Much of the achieved advances result from the development and availability of instrumentation to study slow and fast reactions at pressures up to 300 MPa, including stopped-flow, T-jump, P-jump, NMR, ESR, flash-photolysis, and pulse-radiolysis instrumentation (1, 2, 4, 6, 7). Readers are advised to consult the quoted references for more detailed information, since these present a detailed account of the present instrumentation and commercial availability of such equipment. 

The systems that we investigated in collaboration with others involved intermolecular and intramolecular electron-transfer reactions between ruthenium complexes and cytochrome c. We also studied a series of intermolecular reactions between chelated cobalt complexes and cytochrome c. A variety of high-pressure experimental techniques, including stopped-flow, flash-photolysis, pulse-radiolysis, and voltammetry, were employed in these investigations. As the following presentation shows, a remarkably good agreement was found between the volume data obtained with the aid of these different techniques, which clearly demonstrates the complementarity of these methods for the study of electron-transfer processes. 

Epr is most effective for detecting free radicals that may occur as intermediates in oxidation and reduction reactions involving transition metal ions. Since these transients are invariably quite labile, epr is combined with continuous flow, (more conveniently) stopped-flow, flash photolysis, and pulse radiolysis. 

Transients such as OJ or HO can be generated in solution by pulse radiolysis of O2. If such solutions are contained in one syringe of a stopped-flow apparatus they may be mixed with substrate and the final mixture examined spectrally. For flow experiments these transients must, of course, have lifetimes longer than a few millisecond. For the examination of more labile transients, production may be by laser photolyses or pulse radiolysis, and the substrate under examination must be then incorporated in the pulsed solution. Care has now to be taken that substantial amounts of the substrate are not lost (by reaction) as a result of the pulse. 

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. 

Rate studies of the reaction between cesium and water in ethylenediamine, using the stopped-flow technique, have been extended to all alkali metals. The earlier rate constant (k — 20 NT1 sec.-1) and, in some cases, a slower second-order process (k — 7 Af"1 sec.-1) have been observed. This is consistent with optical absorption data and agrees with recent results obtained in aqueous pulsed-radiolysis systems. Preliminary studies of the reaction rate of the solvated electron in ethylenediamine with other electron acceptors have been made. The rate constant for the reaction with ethylene-diammonium ions is about 105 NCl sec.-1 Reactions with methanol and with ethanol show rates similar to those with water. In addition, however, the presence of a strongly absorbing intermediate is indicated, which warrants more detailed examination. 

As has already been mentioned, picosecond pulsed radiolysis offers great possibilities for studying the short-lived transient processes. In Ref. 326 the solutions of 2,5-diphenyloxazol (DPO) in different solvents were irradiated by picosecond electron pulses obtained from an accelerator. The authors have found two types of excitations of DPO, which they have named the fast and the slow excitations. With fast excitation the luminescence appears during the electron pulse and stops growing at the end of the pulse, after 10 ps. With slow excitation the luminescence is formed within 1 ns. At small DPO concentrations the observed intensity of fast luminescence cannot be explained by direct excitation by electrons (cf. data of Ref. 325). Analyzing the results of experiments with different solvents and different types of additives, Katsumura et al.326 conclude that the main part of the fast luminescence of DPO is due to VCR absorption. 

The oxidation of phenol by ferrate(V) was studied by use of pre-mix (stopped-flow) pulse radiolysis [15], To avoid complications from the thermal reaction be-... 

Kissner, R., Nauser, T, Bugnon, P, Lye, PG, and Koppenol, WH. 1997. Formation and properties of peroxynitrite as studied by laser flash photolysis, high-pressure stopped-flow technique, and pulse radiolysis. Chem Res Toxicol 10 1285-1292. 

S. Baral, C. Lume-Pereira, E. Janata, A. Henglein (1985). Chemistry of colloidal manganese dioxide. 1. Reaction with O and H2O2 (pulse radiolysis and stop flow studies). J. Phys. Chem., 89, 5779-5783. 

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). 

Transient intermediates are most commonly observed by their absorption (transient absorption spectroscopy see ref. 185 for a compilation of absorption spectra of transient species). Various other methods for creating detectable amounts of reactive intermediates such as stopped flow, pulse radiolysis, temperature or pressure jump have been invented and novel, more informative, techniques for the detection and identification of reactive intermediates have been added, in particular EPR, IR and Raman spectroscopy (Section 3.8), mass spectrometry, electron microscopy and X-ray diffraction. The technique used for detection need not be fast, provided that the time of signal creation can be determined accurately (see Section 3.7.3). For example, the separation of ions in a mass spectrometer (time of flight) or electrons in an electron microscope may require microseconds or longer. Nevertheless, femtosecond time resolution has been achieved,186 187 because the ions or electrons are formed by a pulse of femtosecond duration (1 fs = 10 15 s). Several reports with recommended procedures for nanosecond flash photolysis,137,188-191 ultrafast electron diffraction and microscopy,192 crystallography193 and pump probe absorption spectroscopy194,195 are available and a general treatise on ultrafast intense laser chemistry is in preparation by IUPAC. 

In 1993 Weiss, Riley, and co-workers reported a study on purported SOD mimics by stopped-flow UV-vis spectroscopy (428) in which they assessed reactivity by following the decay of the superoxide absorption at 245 nm. Two of the earlier techniques that had been used to assess SOD activity included observation by UV-vis spectroscopy of the oxidation of nitroblue tetrazolium (NBT) (68) or the oxidation of a cytochrome c by superoxide (52). Both systems used superoxide from an in situ generator, frequently xanthine oxidase, wherein the complex being analyzed was compared to a calibrated oxidation of the chromophore alone and in the presence of MnSOD. The direct observation of the decrease in the superoxide signal with time by UV-vis is also possible, and superoxide may be introduced as a solution (428) or generated, in some cases, by pulsed radiolysis (79, 80). In these direct observation experiments, the rate of decay of superoxide in the presence of the complex is compared to the rates of decay of superoxide alone and in the presence of one unit of activity of MnSOD. In all cases, the systems are usually referenced, or calibrated, against the same set of conditions with MnSOD. Due to interactions with cytochrome c with components of assay mixtures other than superoxide, false readings of activity were often observed for some early SOD mimics. The NBT, stopped-flow, or pulsed radiolysis techniques have tended to provide the more accurate answers on the ability of reputed MnSOD mimics. To be considered active in any manner with respect to the decay of superoxide in the stopped-flow analyses, Weiss et al. stated that compounds based on their analyses needed to exhibit kcat values in excess of 10B 5 M 1 s 1 (428). 

In general, a self-exchange rate is best estimated from very low driving force reactions involving similar reaction partners. Furthermore, the Os(V) is unstable toward Os(III), which renders apphcation of more conventional techniques, such as stopped flow, difficult. Accordingly, we used pulse radiolysis to produce Os(V),... 

Synthetic Control as a Probe of Mechanism. In certain systems, synthetic control of very subtle power is possible. The oxidation of water to dioxygen by Ru(bpy)3 has long (15-17) been of interest because this process is one putative step in the use of Ru(bpy)3 to mediate the photodecomposition of water into its elements. In the reaction of Ru(bpy)3 with water/hydroxide ion, electron transfer to produce Ru(bpy)3 + OH can be postulated as an elementaiy step. The OH would be expected to add rapidly to the aromatic ring to yield Ru(bpy)2(bpyOH). Studies of the Ru(bpy)3 + OH- reaction by stopped-flow techniques reveal an intermediate absorbing at long wavelengths. To test whether this species might be the OH-radical adduct, pulse radiolysis was used to study the reaction of OH with Ru(bpy)3. ... 

T he observation by optical spectroscopy of transient species formed in short bursts of radiation requires a reasonable number of these transients to be formed. For the pulse radiolysis of liquids the dose per pulse required is 1-10 Krad. delivered in about 0.1 sec. At a pressure of a few atmospheres, however, the stopping power of gases per unit volume is small and to produce a reasonable number of transient species, a much higher dose per pulse is required. 

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