Alkyl free radicals from the beta-scission of fatty acid alkoxyl radicals as detected by spin trapping in a lipoxygenase system

2-Methyl-2-nitrosopropane (tNB)-radical adducts from incubation mixtures of fatty acids and soybean lipoxygenase in borate buffer (pH 9.0) were measured by electron paramagnetic resonance (EPR). In addition to the previously reported six-line signal of secondary carbon-centered radicals (RCHR'), a weak signal submerged in the baseline was detected after the peroxidation phase was finished. We propose that this radical is a decomposition product formed via beta-scission of fatty acid alkoxyl radicals. EPR spectra of tNB-radical adducts formed in mixtures of either linoleic acid, arachidonic acid, or 15-hydroperoxyeicosatetraenoic acid with lipoxygenase exhibited hyperfine structure characteristic of tNB/.CH2CH2-R with hyperfine coupling constants: aN = 17.1 G; aH beta = 11.2 G (2H); and aH gamma = 0.6 G (2H). In the case of linolenic acid, this radical tNB/.CH=CH-R' with hyperfine coupling constants: aN = 17.1 G; aH beta = 10.9 G (2H); aH gamma = 1.1 G; and aH delta = 0.5 G. In accord with the decomposition scheme of hydroperoxides derived from unsaturated fatty acids, the radical adducts tNB/.CH2CH2-R and tNB/.CH2-CH=CH-R' were assigned as the pentyl and 2-pentenyl radicals, respectively.     

An ESR study of radical kinetics in L-alpha-amino-n-butyric acid hydrochloride containing L-cysteine hydrochloride

On annealing at temperatures near 100 degrees C, carbon-centered radicals migrate to sulfur-centered radicals in X-irradiated crystals of L-alpha-amino-n-butyric acid hydrochloride, CH3CH2CH(NH3-Cl)COOH, containing L-cysteine hydrochloride, SHCH2CH(NH3Cl)COOH. Samples containing 0, 0.5, 1.0, and 1.5% L-cysteine hydrochloride were studied. When no cysteine is present, the carbon-centered radical formed by X irradiation, CH3CH2CHOOH, decays according to a second-order diffusion-controlled rate equation. In samples containing cysteine, the same carbon-centered radicals are formed, but on annealing, they migrate to cysteine, where a perithiyl radical, RSS, is formed. The transfer of carbon-centered radicals to perthiyl radicals follows a pseudo first-order rate equation with an activation energy of 1.15 eV. A decrease in the initial concentration of the carbon-centered radicals or an increase in the initial concentration of cysteine results in an increase in the transfer efficiency. The rate of growth of the perthiyl radical depends on both the initial concentration of cysteine and the initial concentration of carbon-centered radicals. The pseudo first-order rate constant increases when either the initial carbon-centered radical concentration increases or the initial cysteine concentration increases. The mechanism by which radicals move from one lattice site to another in the crystalline material is most likely hydrogen abstraction from a neighboring molecule.     

The reactions of the hydroxymethyl radical with 1,3-dimethyluracil and 1,3-dimethylthymine

Hydroxymethyl radicals .CH2OH, generated by the radiolysis of methanol (0.5 mol dm-3) in N2O-saturated aqueous solutions, were reacted with 1,3-dimethyluracil or 1,3-dimethylthymine (10(-3) mol dm-3). The products were identified and their G values determined. It has been concluded that in 1,3-dimethyluracil .CH2OH attack occurs only at C(6) while in 1,3-dimethylthymine there is partitioning between addition (two-thirds) and H-abstraction from the C(5)-methyl group (one-third). A rate constant for CH2OH addition to 1,3-dimethyluracil of about 10(4) dm3 mol-1 s-1 is estimated. Complexities that may arise in the radiolysis of pyrimidines such as 1,3-dimethylthymine, apparently as a consequence of the formation of 5-alkylidenepyrimidines, are discussed. A value of 0.15 has been estimated for the disproportionation/combination ratio for the hydroxymethyl radical self-termination reaction.     

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO(.-) and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO(.-)), it forms methyl radicals ((.-)CH(3)) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO(.-) formation. However, we recently reported that (.-)CH(3) might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl 1]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO(.-) and DMSO. In addition to identifying the two well-known free radicals, (.-)CH(3) and (.-)OCH(3), from this interaction, we also characterized two additional free radicals, (.-)CH(2)OH and (.-)CH(2)S(O)CH(3). Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/(.-)CH(3) was not detected. However, with the addition of the LC/MS technique, we found approximately 0.75 microM POBN/(.-)CH(3) hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.     

Radiation-induced free radicals in solid 5-halouracil derivatives: single crystals of 5-iododeoxyuridine

X-irradiation of single crystals of 5-iododeoxyuridine (IUdR) in the temperature range 8-300 K produces mainly four different radicals which have been studied by electron spin resonance (e.s.r.) and electron nuclear double resonance (ENDOR)-spectroscopy. At low temperatures, a pi-anion is formed which shows predominantly an interaction of the unpaired electron with a proton at carbon C6 of the base (-11.8 G, -23.9 G, -4.6 G). Above 10-20 K, the anion protonates at C6 to yield a RC-I(CH2)-R' radical comprising alpha-iodo and beta-methylene proton hyperfine interactions. The primary oxidation product is an O5'-situated alkoxy radical RCH2O which shows inequivalent beta-proton couplings of about 100 G and 35 G together with a highly anisotropic g-tensor. Upon warming to 265 K, a C2'-located radical on the deoxyribose is formed which is stable at room temperature. A detailed account of its spectral features as obtained by ENDOR exhibits three different alpha-type couplings, two small beta-protons and a dipolar interaction. Other radicals, not reproducibly observed, involve a C5'-hydroxyalkyl radical and a species related to the base cation at low temperatures.     

EPR detection of lipid-derived free radicals from PUFA, LDL, and cell oxidations

We have used the spin trap 5,5-dimethyl-pyrroline-1-oxide (DMPO) and EPR to detect lipid-derived radicals (Ld*) during peroxidation of polyunsaturated fatty acids (PUFA), low-density lipoprotein (LDL), and cells (K-562 and MCF-7). All oxygen-centered radical adducts of DMPO from our oxidizable targets have short lifetimes (<20 min). We hypothesized that the short lifetimes of these spin adducts are due in part to their reaction with radicals formed during lipid peroxidation. We proposed that stopping the lipid peroxidation processes by separating oxidation-mediator from oxidation-substrate with an appropriate extraction would stabilize the spin adducts. To test this hypothesis we used ethyl acetate to extract the lipid-derived radical adducts of DMPO (DMPO/Ld*) from an oxidizing docosahexaenioc acid (DHA) solution; Folch extraction was used for LDL and cell experiments. The lifetimes of DMPO spin adducts post-extraction are much longer (>10 h) than the spin adducts detected without extraction. In iron-mediated DHA oxidation we observed three DMPO adducts in the aqueous phase and two in the organic phase. The aqueous phase contains DMPO/HO* aN approximately aH approximately 14.8 G) and two carbon-centered radical adducts (aN1 approximately 15.8 G, aH1 approximately 22.6 G; aN2 approximately 15.2 G, aH2 approximately 18.9 G). The organic phase contains two long-chain lipid radical adducts (aN approximately 13.5 G, aH approximately 10.2 G; and aN approximately 12.8 G; aH approximately 6.85 G, 1.9 G). We conclude that extraction significantly increases the lifetimes of the spin adducts, allowing detection of a variety of lipid-derived radicals by EPR.     

Spin trapping of nitric oxide by aci anions of nitroalkanes.

In alkaline solutions, nitroalkanes (RCH2NO2) undergo deprotonation and rearrange to an aci anion (RHC=NO2-), which may function as a spin trap. Using electron paramagnetic resonance (EPR) spectroscopy, we have investigated suitability of aci anions of a series of nitroalkanes (CH3NO2, CH3CH2NO2, CH3(CH2)2NO2, and CH3(CH2)3NO2) to spin trap nitric oxide (*NO). Based on the observed EPR spectra, the general structure of the adducts, formed by addition of *NO to RHC=NO2-, was identified as nitronitroso dianion radicals of general formula [RC(NO)NO2]*2- in strong base (0.5 M NaOH), and as a mono-anion radical [RCH(NO)NO2]*- in alkaline buffers, pH 10-13. The hyperfine splitting on 14N in the -NO2 moiety (11.2-12.48 G) is distinctly different from the splitting on 14N in the -NO moiety of the adducts (5.23-6.5 G). The structure of the adducts was verified using 15N-labeled *NO, which produced radicals, in which triplet due to splitting on 14N (I = 1) in 14NO/aci nitro adducts was replaced by a doublet due to 15N (I = 1/2) in 15NO/aci nitro adducts. EPR spectra of aci nitromethane/NO adduct recorded in NaOH and NaOD (0.5 M) showed that the hydrogen at alpha-carbon can be exchanged for deuterium, consistent with structures of the adducts being [CH(NO)NO2]*2- and [CD(NO)NO2]*2-, respectively. These results indicate that nitroalkanes could potentially be used as prototypes for development of *NO-specific spin traps suitable for EPR analysis.     

Radioprotective effects of dimethyl sulfoxide in golden hamster embryo cells exposed to gamma rays at 77 K. I. Radical formation as studied by electron spin resonance

Formation of free radicals in golden hamster embryo (GHE) cells in the frozen living state by gamma irradiation has been studied by electron spin resonance spectroscopy at 4.2 and 77 K. The relative yields of H atoms, OH radicals, and organic radicals trapped in the irradiated GHE cells are 12, 72, and 16%, respectively, of total radical yields. When dimethylsulfoxide (DMSO) is added to GHE cells at 77 K, a large quantity of CH2SOCH3 radicals (DMSO radicals) are formed after gamma irradiation. The yields of OH radicals are not affected by the addition of DMSO. When the GHE cell-DMSO mixtures are irradiated with gamma rays at 77 K and then warmed to 111 K, the OH radicals decay, whereas the DMSO radicals do not increase complementarily. Moreover, the decay rates of the OH radicals at 111 K do not depend upon the concentration of DMSO. Thus OH radicals do not react with DMSO during warming of the irradiated sample. When H atoms are produced by gamma irradiation of acid ice at 60 K, the decay rates of the H atoms at 77 K increase with increasing DMSO concentration, indicating that DMSO reacts with H atoms (CH3SOCH3 + H----.CH2SOCH3 + H2) at 77 K by quantum-mechanical tunneling. When the GHE cell-DMSO mixture is irradiated with gamma rays at 77 or 4.2 K in the dark, DMSO ions are produced in addition to DMSO radicals. Therefore it is concluded that DMSO does not scavenge OH radicals, but does capture H atoms, holes and/or electrons in the gamma-irradiated cells, resulting in the remarkable formation of DMSO radicals. This scavenger effect of DMSO may be related to the radioprotection of DMSO against cell killing described in the companion paper (Watanabe et al., Radiat. Res., this issue).     

Reactions of lumiflavin and lumiflavin radicals with .CO2- and alcohol radicals

The kinetics of reaction of oxidized lumiflavin (F0) with the radicals .CO2(-), CH3CHOH, and (CH3)2COH have been investigated at pH 7 and 24 +/- 1 degree C by the pulse radiolysis technique. The radicals have been shown to react with lumiflavin with second-order rate constants of 36 +/- 4, 26 +/- 3, and 20 +/- 3 in units of 10(8) M-1 s-1, respectively. These rate constants are close to the diffusion limit. The main product in each case was the lumiflavin semiquinone radical FH.. By utilization of long pulses (approximately 100 mus), it was shown that the reaction FH. + .AH(alpha) leads to FH- + A(alpha) + H+ [.AH(alpha) = .CO2(-), CH3CHOH, or (CH3)2COH] proceeded for all three types of .AH(alpha) radical with second-order rate constants of 17 (+4,-3), 9 (+5,-3), and 9 (+4,-3), respectively, in the above units. The beta-carbon radical .CH2CH(OH)CH3 added to .FH, forming an alkylated flavin, while the .CH2CH2OH radical appeared to be capable of addition or hydrogen atom donation to .FH.     

A C5'-centred deoxyribose radical in single crystals of 5-chloro-and 5-bromodeoxyuridine X-irradiated at low temperatures.

X-irradiation at 77 K and subsequent warming to 150 K of single crystals of 5-chloro-and 5-bromodeoxyuridine produces a radical located at position C5' of the deoxyribose moiety. The radical exhibits identical spectral properties in both crystal systems. It is characterized by interaction of the unpaired electron with an alpha-proton(-17-0, -8-7, -33-7G), a beta-proton (18-6, 21-3, 17-5 G) as well as an OH-proton (4-8, 7-9, 12-8 G). The principal values of the g-tensor are 2-0034, 2-0049 and 2-0042. The spectral parameters are discussed in relation to those of the same or similar radicals in other nucleosides (-tides).     

EPR and ENDOR study of crystalline cytosine x HCl doped with 5-methylcytosine. Radiation-induced radical formation and hole transfer

Radical formation and hole transfer were investigated in crystals of cytosine.HCl (C.HCl) doped with 0-1.1 mol-% 5-methylcytosine x HCl (5MC x HCl). The doping level was determined by NMR spectroscopy. Crystals and polycrystalline samples were X-irradiated at 295 K, 77 K and 12 K and studied with EPR, ENDOR and FSE spectroscopy at these temperatures. At 295 K the dominant radicals were the so-called 3alphaH radical, formed in 5MC by a net H-abstraction from the methyl group, and the cytosine C6 H-addition (5-yl) radical. At 12 K five radicals were identified. These were the 3alphaH radical, cytosine reduction and oxidation products, and the cytosine C6 and C5 H-addition (5-yl and 6-yl, respectively) radicals. The spectroscopic parameters for the 3alphaH radical are very similar to those of a radical observed previously in the crystalline cytosine derivatives cytidine (CR), 2'deoxycytidine hydrochloride (CdR x HCl), 5'dCMP and 3'CMP as well as in the uracil derivative 2-thiouracil (2-TU). It was shown that amounts of the order of tenths of a percent 5MC x HCl doped into crystals of C.HCl give rise to a considerable yield of 3alphaH radicals after exposure to ionizing radiation both at room temperature and at lower temperatures. This supports a previous suggestion that naturally occurring 5-methylated cytosine impurities may be responsible for the formation of 3alphaH radicals in the crystalline cytosine derivatives CR, CdR.HCl, 5'dCMP and 3'CMP and suggests that the 3alphaH radical in these systems is a 5-methylated base-centered radical. The total radical yield in doped C x HCl crystals increased considerably with the doping level, both at low temperatures and at room temperature, implying that the 3alphaH radical is more stable than the primary cytosine radicals. The relative amounts of the 3alphaH radical were obtained by using simulated benchmark spectra to reconstruct experimental EPR spectra of doped polycrystalline samples. Evidence is presented suggesting that the enhanced yield of the 3alphaH radical in doped samples is due to holes originally formed at cytosine bases and transferred to 5-methylcytosine bases in addition to the 3alphaH radical being less exposed to recombination than other cytosine radicals.     

Studies on reactions of oxidizing sulfur-sulfur three-electron-bond complexes and reducing alpha-amino radicals derived from OH reaction with methionine in aqueous solution

The technique of pulse radiolysis with spectrophotometric detection has been used to investigate the possibility of electron transfer reactions between oxidizing sulfur-sulfur three-electron-bond complexes (Met2/S thereforeS+), or reducing alpha-amino radicals (CH3SCH2CH2CH.NH2) derived from reaction of methionine with OH radicals and hydroxycinnamic acid (HCA) derivatives, riboflavin (RF) or flavin adenine dinucleotide (FAD), respectively. The HCA derivatives, such as caffeic acid, ferulic acid, sinapic acid and chlorogenic acid, widely distributed phenolic acids in fruit and vegetables, have been identified as good antioxidants previously can rapidly and efficiently repair oxidizing three-electron-bond complexes via electron transfer. RF and FAD can oxidize reducing alpha-amino radicals derived from methionine. The electron transfer rate constants approximately 10(9) dm3 x mol(-1)x s(-1) were determined by following the build-up kinetics of species produced.     

Lignin radicals in the plant cell wall probed by Kerr-gated resonance Raman spectroscopy

Lignin radicals are crucial intermediates for lignin biosynthesis in the cell wall of vascular plants. In this work they were for the first time, to our knowledge, selectively observed in wood cell walls by laser-based Kerr-gated resonance Raman spectroscopy, and the observations were supported by density functional theory prediction of their vibrational properties. For dry wood cells a lignin radical Raman band is observed at 1,570 cm(-1) irrespective of species. For wet beech cells they were generated in situ and observed at 1,606 cm(-1). DFT/B3LYP/6-31+G(d) modeling results support that in beech they are formed from syringyl (S) phenolic moieties and in spruce from guaiacyl (G) phenolic moieties. The observed lignin radical band is predicted as G is approximately 1,597 cm(-1) and S is approximately 1,599 cm(-1), respectively, and is assigned the (Wilson notation) nu(8a) phenyl ring mode. The RR band probes lignin radical properties, e.g., spin density distribution, and these respond to charge polarization or hydrogen bonding to proximate water molecules. These observations can be crucial for an understanding of the factors that control cell wall structure during biosynthesis of vascular plants and demonstrate the unique potential of RR spectroscopy of lignin radicals.     

Quantitative 31P NMR detection of oxygen-centered and carbon-centered radical species

Quantitative 31P NMR spin trapping techniques can be used as effective tools for the detection and quantification of many free radical species. Free radicals react with a nitroxide phosphorus compound, 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO), to form stable radical adducts, which are suitably detected and accurately quantified using (31)P NMR in the presence of phosphorus containing internal standards. Initially, the 31P NMR signals for the radical adducts of oxygen-centered (*OH, O2*-) and carbon-centered (*CH3, *CH2OH, CH2*CH2OH) radicals were assigned. Subsequently, the quantitative reliability of the developed technique was demonstrated under a variety of experimental conditions. The 31P NMR chemical shifts for the hydroxyl and superoxide reaction adducts with DIPPMPO were found to be 25.3, 16.9, and 17.1 ppm (in phosphate buffer), respectively. The 31P NMR chemical shifts for *CH3, *CH2OH, *CH(OH)CH3, and *C(O)CH3 spin adducts were 23.1, 22.6, 27.3, and 30.2 ppm, respectively. Overall, this effort forms the foundations for a targeted understanding of the nature, identity, and mechanisms of radical activity in a variety of biomolecular processes.     

Rapid kinetics of tyrosyl radical formation and heme redox state changes in prostaglandin H synthase-1 and -2.

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34-35 G) was formed by 4 ms, followed by transition to a wide singlet (33-34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25-26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.     

Water-soluble Hexasulfobutyl[60]fullerene inhibit low-density lipoprotein oxidation in aqueous and lipophilic phases

Oxidative modification of low-density lipoprotein (LDL) plays a pivotal role in the pathogenesis of atherosclerosis. Increasing the resistance of LDL to oxidation may therefore mitigate, or even prevent, atherosclerosis. A new water-soluble C60 derivative, hexasulfobutyl[60]fullerene [C60 - (CH2CH2CH2CH2-SO3Na)6; FC4S], consisting of 6 sulfobutyl moieties covalently bound onto the C60 cage is a potent free radical scavenger. This study explored the antioxidative effect of sulfobutylated fullerene derivatives (FC4S) on LDL oxidation. FC4S was found to be effective in protecting LDL against oxidation induced by either Cu2+ or azo peroxyl radicals generated initially in the aqueous or lipophilic phase, respectively. Levels of the oxidative products, conjugated diene and thiobarbituric acid-reactive substances, and the relative electrophoresis mobility of the LDL were decreased. The addition of 20 microM FC4S at the early stage of oxidation increased the kinetic lag time from 69 +/- 11 to 14 +/- 10 min (P < 0.05) and decreased the propagation rate from 17.1 +/- 2.6 to 6.3 +/- 1.0 mOD/min (P < 0. 005). Persistent suppression of peroxidation reaction was observed upon further addition of FC4S after full consumption of all endogenous antioxidants during the propagation period. Intravenous injection of hypercholesterolemic rabbits with FC4S (1 mg/kg/day) efficiently decreased atheroma formation. Data substantiate the use of FC4S as an excellent hydrophilic antioxidant in protecting atheroma formation, via removing free radicals, in either aqueous or lipophilic phase.     

Theoretical study of the reactivity properties of two forms of stobadine

The cardio- and neuroprotective effect of the pyridoindole stobadine (S) is conditioned mainly by its good radical scavenging properties. It has been showed by EPR experiment, that the ultimate product of the reaction of stobadine with hydroxyl radical is the nitroxyl radical. However, for the unsaturated dehydrostobadine (DHS) the ultimate product was not experimentally determined, although its reactivity with a hydroxyl radical has been detected. Using the quantum chemical method AM1 we calculated the physico-chemical properties of S, DHS and their radicals. For the stobadine alone, the corresponding radical was formed by removing the H* from the NH group of indol, while in the case of DHS we removed the CH3* from the nitrogen in pyridine ring. For S and DHS we calculated the differences in the energies between the parent molecules and the corresponding radicals as well as the spin distributions for the radicals. The results confirmed the differences in the reactivities of S and DHS.     

Radicals from "Good's" buffers

Oxidative deposition of iron in ferritin or the autoxidation of iron in the absence of protein produces radicals from Good's buffers. Radical species are formed from the piperazine ring-based buffers Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Epps 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, and Pipes 1,4-piperazinediethanesulfonic acid, but not from Mes (4-morpholineethanesulfonic acid) which contains a morpholine ring. The radicals all have half-lives around 10 min and display very similar electron paramagnetic resonance spectra consisting of at least 30 lines. The Hepes radical can be formed by the addition of potassium superoxide directly to the buffer and its production during iron(II) autoxidation is inhibited by superoxide dismutase (EC 1.15.1.1). Catalase (EC 1.11.1.6) accelerates the decay of the EPR spectrum. Thus the buffer radicals are secondary radical species produced from oxygen radicals formed during the iron catalyzed Haber-Weiss process. The deoxyribose/thiobarbituric acid assay for hydroxyl radical production shows that Hepes is an effective hydroxyl radical scavenging agent. The Hepes radical can also be formed electrolytically at a potential of +0.8 V (vs standard hydrogen electrode). Oxidation of Hepes at pH 10 during the autoxidation of iron(II) or by the addition of hydrogen peroxide produces a nitroxide radical. These results indicate that piperazine ring Good buffers should be avoided in studies of redox processes in biochemistry.     

Radical trapping properties of imidazolyl nitrones

The ability of ten imidazolyl nitrones to directly scavenge free radicals (R(*)) generated in polar ((*)OH, O(*)(2)(-), SO(*)(3)(-) cysteinyl, (*)CH(3)) or in apolar (CH(3)-(*)CH-CH(3)) media has been studied. When oxygen or sulfur-centered radicals are generated in polar media, EPR spectra are not or weakly observed with simple spectral features. Strong line intensities and more complicated spectra are observed with the isopropyl radical generated in an apolar medium. Intermediate results are obtained with (*)CH(3) generated in a polar medium. EPR demonstrates the ability of these nitrones to trap radicals to the nitrone C(alpha) atom (alpha radical adduct) and to the imidazol C(5) atom (5-radical adduct). Beside the nucleophilic addition of the radical to the C(alpha) atom, the EPR studies suggest a two-step mechanism for the overall reaction of R(*) attacking the imidazol core. The two steps seem to occur very fast with the (*)OH radical obtained in a polar medium and slower with the isopropyl radical prepared in benzene. In conclusion, imidazolyl nitrones present a high capacity to trap and stabilize carbon-centered radicals.     

Role of glycine-33 and methionine-35 in Alzheimer's amyloid beta-peptide 1-42-associated oxidative stress and neurotoxicity

Recent theoretical calculations predicted that Gly33 of one molecule of amyloid beta-peptide (1-42) (Abeta(1-42)) is attacked by a putative sulfur-based free radical of methionine residue 35 of an adjacent peptide. This would lead to a carbon-centered free radical on Gly33 that would immediately bind oxygen to form a peroxyl free radical. Such peroxyl free radicals could contribute to the reported Abeta(1-42)-induced lipid peroxidation, protein oxidation, and neurotoxicity, all of which are prevented by the chain-breaking antioxidant vitamin E. In the theoretical calculations, it was shown that no other amino acid, only Gly, could undergo such a reaction. To test this prediction we studied the effects of substitution of Gly33 of Abeta(1-42) on protein oxidation and neurotoxicity of hippocampal neurons and free radical formation in synaptosomes and in solution. Gly33 of Abeta(1-42) was substituted by Val (Abeta(1-42G33V)). The substituted peptide showed almost no neuronal toxicity compared to the native Abeta(1-42) as well as significantly lowered levels of oxidized proteins. In addition, synaptosomes subjected to Abeta(1-42G33V) showed considerably lower dichlorofluorescein-dependent fluorescence - a measure of reactive oxygen species (ROS) - in comparison to native Abeta(1-42) treatment. The ability of the peptides to generate ROS was also evaluated by electron paramagnetic resonance (EPR) spin trapping methods using the ultrapure spin trap N-tert-butyl-alpha-phenylnitrone (PBN). While Abeta(1-42) gave a strong mixture of four- and six-line PBN-derived spectra, the intensity of the EPR signal generated by Abeta(1-42G33V) was far less. Finally, the ability of the peptides to form fibrils was evaluated by electron microscopy. Abeta(1-42G33V) does not form fibrils nearly as well as Abeta(1-42) after 48 h of incubation. The results suggest that Gly33 may be a possible site of free radical propagation processes that are initiated on Met35 of Abeta(1-42) and that contribute to the peptide's toxicity in Alzheimer's disease brain.