Radical traps, DMPO

Substrate H2°2+ Reagent an (Gauss) "h (Gauss) DMPO Radical Trapped... 

Anodic Decomposition of Toxic Compounds (Anodic Mineralization), Fig. 4 ESR signals of electrolyzed carbonate solution containing 20 mM phenol. The number is elapsed times (min) after the addition of DMPO (radical trapping reagent) to the electrolyte extracted from the cell... 

Work by Harbour, Chow and Bolton (1974) on the spin adducts of superoxide (or HOO )13 with nitrones paved the way for a number of investigations of superoxide and hydroperoxyl radical chemistry. Harbour and Bolton (1975) used DMPO to trap superoxide formed by spinach chloroplasts in the presence of 02. The signal strength was greatly enhanced when methylviologen was present, consistent with the hypothesis that this bis-pyridinium dication accepts an electron from the primary acceptor of photoprotein I, and then transfers it to molecular oxygen. 

Thus there is little doubt that the hydroxyl radical, if generated by an unambiguous method such as pulse radiolysis, can be trapped by PBN or DMPO, even if the former has several deficiencies, among them low trapping efficiency and short half-life of HO-PBN. The problem in hydroxyl radical trapping thus rests with the possible competition from the nucleophilic addition-oxidation mechanism, as exemplified in reaction (69) for DMPO and Ox-Red as a general one-electron redox system, or the inverted spin trapping mechanism (70). The treatment to follow will mostly be limited to DMPO. 

The radical addition products are nitroxyl radicals which are readily characterized by means of their EPR spectra. We used both radical trapping agents to carry out some of the studies. The PBN system is less diagnostic because it is not as sensitive to trapping all the radical species as DMPO. The data in Table II are for the DMPO trap. We also generated hydroxyl... 

The sequence of events following the reaction of metMb with hydrogen peroxide has also been investigated through the use of spin trapping agents. Initial studies of this type with 5,5-dimethylpyrroline N-oxide (DMPO) led to the identification of Y103 as the primary site of DMPO adduct formation, a modification that was blocked by specific iodination of this residue 187). The identity of the radical trapped in this reaction... 

In the vascular system, a particularly important fate of peroxynitrite may be its rapid reaction with Hb-Fe(II)-02. Studies by Romero et al. have led to the proposal of a mechanism for the formation of Hb-Fe(III), involving the displacement of 02 - from Hb-Fe(TI)-02 by ONOO, with the formation of Hb-Fe(III)-ONOO. This transient decays mainly to NO and Hb-Fe(III), but ca. 10 % gives Hb-Fe(IV) = 0 plus N02. Tyrosyl and cysteinyl radicals, trapped using MNP and DMPO, respectively, were proposed to arise via electron transfer from the protein moiety to the ferryl haem.97 In an earlier study, a mechanism involving N02 generation had been suggested for the formation of Hb-Fe(IV) = 0 from Hb-Fe(II)-02 by ONOOH.98... 

Fig. 6.14 Experimental evidence for the formation of OH radicals generated in photo-initiated AOPs mechanism of the DMPO spin trapping technique.

Sun L, Hoy AR, Bolton JR (1996) Generation Efficiency of the Hydroxyl Radical Adduct of the DMPO Spin Trap in Homogeneous and Heterogeneous Media, J Adv. Oxid. Technol. 1, No. 1 44—52... 

The much studied reaction of Fe"EDTA with HjOj has been investigated in a stopped-flow system with a deadtime of 18 ms using 5,5-dimethyl-1-pyrroline N-oxide, DMPO, to trap the OH radicals. The general reactions are shown in Scheme 10.7. 

Zhang, X., Wang, H and Guo, Y. (2006) Interception of the radicals produced in electrophilic fluorination with radical traps (TEMPO, DMPO) studied by electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom., 20, 1877-1882,... 

Spin Trap Synthesis and purification -Octanoljwater partition coefficient Examples of radicals trapped Lifetime of superoxide radical adduct relative to DMPO Comments... 

Nonsupported metal black catalysts were then used to eliminate the strong ESR signal from the carbon support. No radicals could be detected for perfluorobutane sulfonate nor for the Nafion ionomer dispersion presumably owing to the short lifetime of the radicals (Vogel et al. 2(X)7). With a DMPO spin trap, HO adduct can be easily observed in Fenton s test, while a small amount of HO, can also be detected. 

By determination of the parameters of the spin adduct spectrum it is often possible to identify the nature of the primary trapped radical, or at least to determine the type of radical trapped. Table 1 gives examples of the range of hyperfine coupling constants obtained for a variety of trapped species for the two most commonly used spin traps DMPO (dimethyl-pyridine N-oxide) and PBN (a-phenyl-N-t-butyl nitrone). Care has to be taken, however, because the coupling constants vary with solvent polarity (for example, in water the DMPO adduct of the t-butoxy radical has < (N) = 1.48 and fl(H) = 1.60 mT while in toluene < (N) = 1.3 and fl(H) = 0.75 mT). 

The formation of ROS was quantified using trapping ejqjeriments in which synthetic melanin films are treated with various metal salt solutions. After equilibration and extensive washing, the sanq)les were allowed to auto-oxidize under air in the presence of the radical trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Electron paramagnetic resonance characterization of the trapped ROS showed an distinctive increase in melanin s auto-oxidation upon metal-ion binding.(25) The increase in HO radical trapped by DMPO after treating the melanin with solutions of Zn(II) and Cu(II) salts correlated with the increase in quinone imine concentrations predicted. 

Using the same experimental conditions, we measured hydroxyl radical formation, using radical trap DMPO, for both DHI-melanin and melanoma cells in culture.(25) The con arison was telling for both, addition of CAT decreased the signal, while SOD caused a large increase in the hydroxyl-radical adduct. Our hypothesis is that the pro-oxidant response in both chemical and cell culture studies are due to the pro-oxidant reactivity of melanin. 

Likewise, photochemical reactions of N-oxides are considered in the following, not the use of N-oxides in photochemistry. Therefore, the use of nitrones, in particular of cyclic polysubstituted cyclic nitrones such as 5,5-dimethylpyrroline N-oxide (DMPO, 8, see Equation 99.4), as radical traps for oxygen-centered radicals, in particular the hydroxyl radical or the superoxide anion,is not discussed here. In fact, these nitrones are used for determining the mechanism of photoinduced oxidation reactions, for example, in lipid photooxidation " or in oxidative DNA damage but are not undergoing a photochemical reaction under such circumstances. 

Fig. 4.21 EPR spectra of radicals trapped by DMPO in P25 and P25 + 0.5 % GR dispersions, (a) DMPO-O2 formed in irradiated methanol dispersions (b) DMPO- OH framed in irradiated aqueous dispersions (Reprinted with permission from Ref. [73] Copyright 2010, American Chemical Society), (c) and (d), EPR spectra of superoxide radical species trapped by DMPO in Ti02-5 % GR and Ti02-5 % CNT dispersions in BTF solvent under visible light irradiation (Reprinted with permission from Ref. [5] Copyright 2011, American Chemical Society)...

ESR studies on the initial free radicals were carried out by using MNP(2-methyl-2-nitrosopropane) or DMPO (5,5-dimethylpyrroline N-oxide) as the spin-trapping agent. The reactions are shown as ... 

In related work, the reactions of hydrogen peroxide with iron(II) complexes, including Feu(edta), were examined.3 Some experiments were carried out with added 5.5"-dimethyl-1-pyrroline-N-oxide (DMPO) as a trapping reagent fa so-called spin trap) for HO. These experiments were done to learn whether HO was truly as free as it is when generated photochemically. The hydroxyl radical adduct was indeed detected. but for some (not all) iron complexes evidence was obtained for an additional oxidizing intermediate, presumably an oxo-iron complex. 

A spin trap is a diamagnetic compound that reacts with a radical by addition of the radical functionality typically to a double bond in the trap, thus forming a new radical that is more stable (better, less unstable) than the original radical. By far the most common class of spin traps are nitrone compounds that, upon addition of the primary radical, produce a stable aminoxyl radical (Figure 10.1). The compound DMPO is the paradigmatic spin trap it is readily available, widely used, and its EPR spectra are relatively easy to interpret. Some of its radical adducts have unpractically short lifetimes. 

Thus, superoxide itself is obviously too inert to be a direct initiator of lipid peroxidation. However, it may be converted into some reactive species in superoxide-dependent oxidative processes. It has been suggested that superoxide can initiate lipid peroxidation by reducing ferric into ferrous iron, which is able to catalyze the formation of free hydroxyl radicals via the Fenton reaction. The possibility of hydroxyl-initiated lipid peroxidation was considered in earlier studies. For example, Lai and Piette [8] identified hydroxyl radicals in NADPH-dependent microsomal lipid peroxidation by EPR spectroscopy using the spin-trapping agents DMPO and phenyl-tcrt-butylnitrone. They proposed that hydroxyl radicals are generated by the Fenton reaction between ferrous ions and hydrogen peroxide formed by the dismutation of superoxide. Later on, the formation of hydroxyl radicals was shown in the oxidation of NADPH catalyzed by microsomal NADPH-cytochrome P-450 reductase [9,10]. 

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