The SO4(.-)-induced chain reaction of 1,3-dimethyluracil with peroxodisulphate

The sulphate radical SO4(.-) reacts with 1,3-dimethyluracil (1,3-DMU) (k = 5 X 10(9) dm3 mol-1 s-1) thereby forming with greater than or equal to 90 per cent yield the 1,3-DMU C(5)-OH adduct radical 4 as evidenced by its absorption spectrum and its reactivity toward tetranitromethane. Pulse-conductometric experiments have shown that a 1,3-DMU-SO4(.-) aduct 3 as well as the 1,3-DMU radical cation 1, if formed, must be very short-lived (t1/2 less than or equal to 1 microsecond). The 1,3-DMU C(5)-OH adduct 4 reacts slowly with peroxodisulphate (k = 2.1 X 10(5) dm3 mol-1 s-1). It is suggested that the observed new species is the 1,3-DMU-5-OH-6-SO4(.-) radical 7. At low dose rates a chain reaction is observed. The product of this chain reaction is the cis-5,6-dihydro-5,6-dihydroxy-1,3-dimethyluracil 2. At a dose rate of 2.8 X 10(-3) Gys-1 a G value of approximately 200 was observed ([1,3-DMU] = 5 X 10(-3) mol dm-3; [S2O8(2-)] = 10(-2) mol dm-3; [t-butanol] = 10(-2) mol dm-3). The peculiarities of this chain reaction (strong effect of [1,3-DMU], smaller effect of [S2O(2-)8]) is explained by 7 being an important chain carrier. It is proposed that 7 reacts with 1,3-DMU by electron transfer, albeit more slowly (k approximately 1.2 X 10(4) dm3 mol-1 s-1) than does SO4(.-). The resulting sulphate 6 is considered to hydrolyse into 2 and sulphuric acid which is formed in amounts equivalent to those of 2. Computer simulations provide support for the proposed mechanism. The results of some SCF calculations on the electron distribution in the radical cations derived from uracil and 1-methyluracil are also presented.     

Spin-trapping of superoxide ion by a water-soluble, nitroso-aromatic spin-trap

Spin-trapping of superoxide ion, O2-, which is produced from two different sources (OH(-)-DMSO and xanthine-xanthine oxidase systems), was investigated by use of a water-soluble, notroso-aromatic spin trap, sodium 3,5-dibromo-4-nitrosobenzene-sulfonate (DBNBS). It was found that O2- from all sources was easily trapped by DBNBS to yield the stable O2- adduct showing the ESR spectrum consisting of a triplet of a triplet [aN (1) = 12.63 G and aH (2) = 0.71 G]. Hydroperoxy radical (HO2.), which can be generated from the oxidation of hydrogen peroxide with Ce4+ ion, was not trapped by DBNBS. These results indicate that the trapped radical is O2-, but not HO2..     

Phosphate ester cleavage in ribose-5-phosphate induced by OH radicals in deoxygenated aqueous solution. The effect of Fe(II) and Fe(III) ions.

The reaction of OH radicals and H atoms with ribose-5-phosphate (10(-2) M) in deoxygenated aqueous solution at room temperature (dose-rate 2-1 X 10(17) eV/ml-min, dose 5 X 10(18)-15 X 10(18) eV/ml) leads to the following dephosphorylation products (G-values): ribo-pentodialdose 1 (0-2), 2-hydroxy-4-oxoglutaraldehyde 2 (0-06), 5-deoxy-erythro-pentos-4-ulose 3 (0-1) and 3-oxoglutaraldehyde 4 (0-06). In addition, some minor phosphate free products (total G=0-09) are formed. G(inorganic phosphate) =1-3 and G(H2O2)=0-3. On the addition of 10(-3) M (Fe(III) ions, G (1) and G (3) increase to 0-6 and 0-4 respectively. In the presence of 10(-3) M Fe(II), G(1) and G(3) change to 0-4 and 0-8, respectively. The other dephosphorylation products are suppressed by the iron ions. G(1) also increases on the addition of increasing amounts of H2O2. Each product can be assigned a precursor radical formed by hydrogen abstraction from C-5, C-4 or C-3 of the ribose-5-phosphate molecule. Products 1 and 2 are formed by oxydative dephosphorylation of an alpha-phospho radical with preceeding H2O elimination for product 2. Elimination of H3PO4 from a beta-phospho radical leads to product 3; product 4 is formed by elimination of two molecules of H2O from its precursor radical and hydrolytic cleavage of an enol phosphate bond. Deuterium-labelling experiments and the effects of the iron ions and of H2O2 support the mechanisms proposed. The importance of the dephosphorylation mechanisms for the formation of strand breaks in DNA is discussed with special reference to the effects of the radiosensitizers.     

Monte Carlo calculation of the primary radical and molecular yields of liquid water radiolysis in the linear energy transfer range 0.3-6.5 keV/micrometer: application to 137Cs gamma rays

Monte Carlo simulations of the radiolysis of neutral liquid water and 0.4 M H(2)SO(4) aqueous solutions at ambient temperature are used to calculate the variations of the primary radical and molecular yields (at 10(-6)s) as a function of linear energy transfer (LET) in the range approximately 0.3 to 6.5 keV/micrometer. The early energy deposition is approximated by considering short (approximately 20-100 micrometer) high-energy (approximately 300-6.6 MeV) proton track segments, over which the LET remains essentially constant. The subsequent nonhomogeneous chemical evolution of the reactive species formed in these tracks is simulated by using the independent reaction times approximation, which has previously been used successfully to model the radiolysis of water under various conditions. The results obtained are in good general agreement with available experimental data over the whole LET range studied. After normalization of our computed yields relative to the standard radical and molecular yields for (60)Co gamma radiation (average LET approximately 0.3 keV/micrometer), we obtain empirical relationships of the primary radiolytic yields as a function of LET over the LET range studied. Such relationships are of practical interest since they allow us to predict a priori values of the radical and molecular yields for any radiation from the knowledge of the average LET of this radiation only. As an application, we determine the corresponding yields for the case of (137)Cs gamma radiation. For this purpose, we use the value of approximately 0.91 keV/micrometer for the average LET of (137)Cs gamma rays, chosen so that our calculated yield G(Fe(3+)) for ferrous-ion oxidation in air-saturated 0.4 M sulfuric acid reproduces the value of 15.3 molecules/100 eV for this radiation recommended by the International Commission on Radiation Units and Measurements. The uncertainty range on those primary radical and molecular yields are also determined knowing the experimental error (approximately 2%) for the measured G(Fe(3+)) value. The following values (expressed in molecules/100 eV) are obtained: (1) for neutral water: G(e(-)(aq)) = 2.50 +/- 0.16, G(H(.)) = 0.621 +/- 0.019, G(H(2)) = 0.474 +/- 0.025, G((.)OH) = 2.67 +/- 0.14, G(H(2)O(2)) = 0.713 +/- 0.031, and G(-H(2)O) = 4.08 +/- 0.22; and (2) for 0.4 M H(2)SO(4) aqueous solutions: G(H(.)) = 3.61 +/- 0.09, G(H(2)) = 0.420 +/- 0.019, G((.)OH) = 2.78 +/- 0.12, G(H(2)O(2)) = 0.839 +/- 0.037, and G(-H(2)O) = 4.46 +/- 0.16. These computed values are found to differ from the standard yields for (60)Co gamma rays by up to approximately 6%.     

A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide

The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N(2)- and O(2)-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H(2)O(2) were calculated using hyperfine coupling constants of a(H2,6) = 1.3 G and a(H3,5) = 7.0 G for the ring and a(beta)(H1) = 16.7 G and a(beta)(H2) = 14.2 G for the methylene protons, and are consistent with a highly constrained, conformationally unstable tyrosyl radical. Spectra obtained at later time points contained a mixture of the 3 s signal and another signal that was insufficiently resolved for simulation. Efficient spin trapping with 3, 5-dibromo-4-nitrosobenzenesulfonic acid was observed only when the spin trap was present at the time of H(2)O(2) addition. Spin trapping experiments with either 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or perdeuterated 2-methyl-2-nitrosopropane (MNP-d(9)), which have been shown to trap tyrosyl radicals, were nearly equally effective when the spin trap was added before or 10 min after the addition of H(2)O(2). The superhyperfine structure of the ESR spectra obtained from Pronase-treated MNP-d(9)/*metMb confirmed the assignment to a tyrosyl radical. Delayed spin trapping experiments with site-directed mutant myoglobins in which either Tyr-103 or Tyr-146 was replaced by phenylalanine indicated that radical adduct formation with either DMPO or MNP-d(9) requires the presence of Tyr-103 at all time points, implicating that residue as the radical site.     

Reactivity and activation of dioxygen-derived species in aprotic media (a model matrix for biomembranes)

In aprotic media the electrochemical reduction of dioxygen yields superoxide ion (O2-), which is an effective Br?nsted base, nucleophile, one-electron reductant, and one-electron oxidant of reduced transition metal ions. With electrophilic substrates (organic halides and carbonyl carbons) O2- displaces a leaving group to form a peroxy radical (ROO.) in the primary process. Superoxide ion oxidizes the activated hydrogen atoms of ascorbic acid, catechols, hydrophenazines and hydroflavins. Combination of O2- with 1,2-diphenylhydrazine yields the anion radical of azobenzene, which reacts with O2 to give azobenzene and O2- (an example of O2--induced autoxidation). With phenylhydrazine, O2- produces phenyl radicals. The in situ formation of HO2. (O2- plus a proton source) results in H-atom abstraction from allylic and other groups with weak heteroatom--H bonds (binding energy (b.e.) less than 335 kJ). This is a competitive process with the facile second-order disproportionation of HO2. to H2O2 and O2 (kbi approximately equal to 10(4) mol-1 s-1 in Me2SO). Addition of [FeII(MeCN)4] (ClO4)2 to solutions of hydrogen peroxide in dry acetonitrile catalyses a rapid disproportionation of H2O2 via the initial formation of an adduct [FeII(H2O2)2+----Fe(O)(H2O)2+], which oxidizes a second H2O2 to oxygen. In the presence of organic substrates such as 1,4-cyclohexadiene, 1,2-diphenylhydrazine, catechols and thiols the FeII-H2O2/MeCN system yields dehydrogenated products; with alcohols, aldehydes, methylstyrene, thioethers, sulphoxides, and phosphines, the FeII(H2O2)2+ adduct promotes their monoxygenation. The product from the FeO2+-H2O2 reaction, [FeII(H2O2)22+], exhibits chemistry that is closely similar to that for singlet oxygen (1O2), which has been confirmed by the stoichiometric dioxygenation of diphenylisobenzofuran, 9,10-diphenylanthracene, rubrene and electron-rich unsaturated carbon-carbon bonds (Ph2C = CPh2, PhC = CPh and cis-PhCH = CHPh). In dry ligand-free acetonitrile (MeCN), anhydrous ferric chloride (FeIIICl3) activates hydrogen peroxide for the efficient epoxidation of alkenes. The FeIIICl3 further catalyses the dimerization of the resulting epoxides to dioxanes. These observations indicate that strong Lewis acids that are coordinatively unsaturated, [FeII(MeCN)4]2+ and [FeIIICl3], activate H2O2 to form an effective oxygenation and dehydrogenation agent.(ABSTRACT TRUNCATED AT 400 WORDS)     

A time-resolved electron spin resonance study of the oxidation of ascorbic acid by hydroxyl radical.

Time-resolved electron spin resonance (ESR) spectroscopy for the study of radicals produced by pulse radiolysis is illustrated by a study of the oxidation of ascorbic acid by OH radical in aqueous solution. In basic solution, the direct oxidation product, the ascorbate mono-anion radical, is formed within less than 2 mus of the radiolysis pulse. In acid solutions (pH 3(-4.5), N(2)O:saturated) three radicals are initially formed, the ascorbate mono-anion radical, an OH adduct seen also in steady-state ESR experiments, and an OH adduct at C2 with the main spin density at C3 of the ring. The first OH adduct decays with an initial half-life of about 100 mus, probably by biomolecular reaction. The second OH adduct, which shows one hyperfine splitting about a(H) = 24.4 +/- 0.3 G and g = 2.0031 +/- 0.0002, decays with a half-life of about 10 mus. On this same time scale the concentration of the ascorbate radical approximately doubles. It is concluded that the adduct at C2, but not the other adduct, loses water rapidly to form the ascorbate radical.     

Electron spin resonance study of free radicals formed from a procyanidin-rich pine (Pinus maritima) bark extract, pycnogenol

The free radical generated from the oxidation of a French maritima pine bark extract Pycnogenol (PYC), by the horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) system at pH 7.4-10.0 was studied using electron spin resonance (ESR) spectrometer. The formation rate of the PYC radical (aH = 0.92 G; g = 2.0055) was dependent on the PYC and HRP concentrations and pH; the lifetime of the radical was up to 90 min. Furthermore, it was found that the PYC radical was mainly composed of the secondary radical formed from procyanidin B3, one of major procyanidins in PYC. The primary radical signal of procyanidin B3 with hyperfine splitting constants aH = 3.67 G (1H), aH = 0.92 G (3H), and g = 2.0055 was transient and disappeared quickly, whereas its secondary radical signal appeared and increased with time. The secondary radical from dimer procyanidin B3 showed quite high stability, differing from the radical from monomer (+)-catechin that could not be observed possibly because of its instability. These results provide evidence to support the idea that the intramolecular hydrogen bond between the O* at the 4' position in one B ring and an OH group in the other B ring of procyanidin B3 is formed during its oxidation in the presence of HRP and H2O2.     

Chlorido-, aqua-, 9-ethylguanine- and 9-ethyladenine-adducts of cytotoxic ruthenium arene complexes containing O,O-chelating ligands

The synthesis and X-ray structures of a half-sandwich Ru(II)p-cymene beta-diketonato complex as chlorido-, aqua-, 9-ethylguanine- and 9-ethyladenine-adducts are reported. Structural features which contribute to stabilisation of adducts through non-covalent, weak interactions are discussed. The X-ray crystal structure of the cytotoxic complex [(eta(6)-p-cym)Ru(Ph(2)acac)Cl] (1), where Ph(2)acac=1,3-diphenyl-1,3-propanedionate and p-cym=para-cymene, shows that the phenyl rings of the acac-type ligand form a hydrophobic face, conferring lipophilic character on the complex. The structure of the aqua adduct [(eta(6)-p-cym)Ru(Ph(2)acac)H(2)O]CF(3)SO(3).H(2)O.Et(2)O (4.H(2)O.Et(2)O), a possible activated species, possesses a comparatively short Ru-OH(2) bond. In the structure of [(eta(6)-p-cym)Ru(Ph(2)acac)9EtG-N7]CF(3)SO(3).2tol (5.2tol), where tol=toluene and 9EtG=9-ethylguanine, a comparatively long Ru-N7 bond is observed in addition to weak G CH8cdots, three dots, centeredO (Ph(2)acac) H-bonds. The crystal structure of [(eta(6)-p-cym)Ru(acac)9EtA-N7]PF(6) (6), where acac=acetylacetonate and 9EtA=9-ethyladenine, a rare example of a ruthenium complex containing monodentate adenine, shows a strong H-bonding interaction between N6Hcdots, three dots, centeredO(acac), which may contribute to the selectivity of {(eta(6)-p-cym)Ru(acac)}(+) towards adenine bases.     

Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical

The effect of bicarbonate anion (HCO(3)(-)) on the peroxidase activity of copper, zinc superoxide dismutase (SOD1) was investigated using three structurally different probes: 5, 5'-dimethyl-1-pyrroline N-oxide (DMPO), tyrosine, and 2, 2'-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS). Results indicate that HCO(3)(-) enhanced SOD/H(2)O(2)-dependent (i) hydroxylation of DMPO to DMPO-OH as measured by electron spin resonance, (ii) oxidation and nitration of tyrosine to dityrosine, nitrotyrosine, and nitrodityrosine as measured by high pressure liquid chromatography, and (iii) oxidation of ABTS to the ABTS cation radical as measured by UV-visible spectroscopy. Using oxygen-17-labeled water, it was determined that the oxygen atom present in the DMPO-OH adduct originated from H(2)O and not from H(2)O(2). This result proves that neither free hydroxyl radical nor enzyme-bound hydroxyl radical was involved in the hydroxylation of DMPO. We postulate that HCO(3)(-) enhances SOD1 peroxidase activity via formation of a putative carbonate radical anion. This new and different perspective on HCO(3)(-)-mediated oxidative reactions of SOD1 may help us understand the free radical mechanism of SOD1 and related mutants linked to amyotrophic lateral sclerosis.     

Acute methanol intoxication generates free radicals in rats: an ESR spin trapping investigation

Electron spin resonance (ESR) spectroscopy has been used to investigate free radical generation in rats with acute methanol poisoning. The spin trapping technique was used where a spin trapping agent, alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), reacted with the corresponding alcohol-derived or alcohol-dependent radical to form radical adducts. One radical adduct was detected in both bile and urine samples 2 h after acute methanol poisoning in male Sprague Dawley rats. The hyperfine coupling constants for the radical adduct from [(13)C]-labeled methanol detected in the bile were a(N) = 15.58, a(beta)(H) = 2.81 G, and a(beta)(13C) = 4.53 G, which unambiguously identified this species as POBN/*CH@OH. The same radical adduct was detected in urine. The identification of a methanol-derived radical adduct in samples from bile and urine provided strong direct evidence for the generation of the alcohol-derived radicals during acute intoxication by methanol. Simultaneous administration of the alcohol dehydrogenase inhibitor 4-methylpyrazole and methanol resulted in an increase in the generation of the free radical metabolite detected in the bile. This is the first ESR evidence of methanol-derived free radical generation in an animal model of acute methanol intoxication.     

The reaction of solvated electrons with cytosine, 5-methyl cytosine and 2'-deoxycytidine in squeous solution. The reaction of the electron adduct intermediates with water, p-nitroacetophenone and oxygen. A pulse spectroscopic and pulse conductometric study.

Using conductivity detection, pulse radiolysis experiments showed that solvent protonation of the electron adducts of cytosine, 5-methyl cytosine and 2'-deoxycytidine occurs with rate constants k greater than or equal to 2 x 10(4) M-1S-1. The protonated electron adducts transfer an electron to p-nitroactetophenone (PNAP) with rate constants ranging from 3.5 x 10(9) to 5.3 x 10(9) M-1S-1. The transfer is quantitative (G = 2.7), as shown by conductometric and spectroscopic measurements. In the presence of O2 no electron transfer to O2 takes place, implying that O2 adds to the protonated electron adduct radicals. No electron transfer from the H- and OH-adducts of the cytosine derivatives, either to PNAP or to O2, takes place near neutral pH. It is suggested that the differences in the reaction behaviour of the H-adduct radicals and the protonated electron adduct radicals towards PNAP can be accounted for if different radicals are formed by H-addition and protonation of the electron adduct. The H atoms most probably add to the C-5-C-6 double bonds, whereas the electron adducts are protonated at N-3 and/or 0-2.     

Clotrimazole inhibits hemoperoxidase of Plasmodium falciparum and induces oxidative stress. Proposed antimalarial mechanism of clotrimazole

The mechanism of antimalarial activity of clotrimazole was studied placing emphasis on its role in inhibiting hemoperoxidase for inducing oxidative stress in Plasmodium falciparum. Clotrimazole, in the presence of H2O2, causes irreversible inactivation of the enzyme, and the inactivation follows pseudo-first order kinetics, consistent with a mechanism-based (suicide) mode. The pseudo-first order kinetic constants are ki = 2.85 microM, k(inact) = 0.9 min(-1), and t(1/2) = 0.77 min. The one-electron oxidation product of clotrimazole has been identified by EPR spectroscopy as the 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) adduct of the nitrogen-centered radical (aN = 15 G), and as DMPO protects against inactivation, this radical is involved in the inactivation process. Binding studies indicate that the clotrimazole oxidation product interacts at the heme moiety, and the heme-clotrimazole adduct has been dissociated from the inactivated enzyme and identified (m/z 1363) by mass analysis. We found that the inhibition of hemoperoxidase increases the accumulation of H2O2 in P. falciparum and causes oxidative stress. Furthermore, the inhibition of hemoperoxidase correlates well with the inhibition of parasite growth. The results described herein indicate that the antimalarial activity of clotrimazole might be due to the inhibition of hemoperoxidase and subsequent development of oxidative stress in P. falciparum.     

A mimic of superoxide dismutase activity protects Chlorella sorokiniana against the toxicity of sulfite

The spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) has been used to demonstrate the light-dependent production of O2- by Chlorella sorokiniana. In the presence of SO3= a light-dependent production of the sulfur trioxy anion radical (SO3-.) could also be seen. A complex prepared by reacting desferrioxamine with MnO2, which catalyzes the dismutation of O2-, protected the alga against the toxicity of sulfite. The data suggest that SO2 toxicity is at least partially due to the effects of sulfoxy-free radicals generated by the oxidation of SO3= by O2-.     

Xanthine oxidase/hydrogen peroxide generates sulfur trioxide anion radical (SO3.-) from sulfite (SO3(2-)).

In the presence of hydrogen peroxide (H2O2), xanthine oxidase has been found to catalyze sulfur trioxide anion radical (SO3.-) formation from sulfite anion (SO3(2-)). The SO3.- radical was identified by ESR (electron spin resonance) spin trapping, utilizing 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) as the spin trap. Inactivated xanthine oxidase does not catalyze SO3.- radical formation, implying a specific role for this enzyme. The initial rate of SO3.- radical formation increases linearly with xanthine oxidase concentration. Together, these observations indicate that the SO3.- generation occurs enzymatically. These results suggest a new property of xanthine oxidase and perhaps also a significant step in the mechanism of sulfite toxicity in cellular systems.     

In vivo singlet-oxygen generation in blood of chromium(VI)-treated mice

Although it is assumed from in vitro experiments that the generation of reactive oxygen species such as the singlet oxygen (1O2), the hydroxyl radical, and the superoxide anion are responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of 1O2 in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 2,2,6,6-tetramethyl-4-piperidone (TMPD), a specific 1O2 trap, to detect 1O2 in blood. The ESR spectrum of the spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:1:1 intensity pattern of three lines with a hyperfine coupling constant A(N) = 16.08 G and a g-value = 2.0066. The concentration of spin adduct detected in the blood was 1.46 microM (0.1% of total Cr concentration). The adduct production was inhibited by the addition of specific 1O2 scavengers such as 1,4-diazabicyclo[2.2.2]octane and sodium azide to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of 1O2 with TMPD. This is the first report of ESR evidence for the in vivo generation of 1O2 in mammals by Cr(VI).     

Kinetics of the interaction of sulfate and hydrogen phosphate radicals with small peptides of glycine, alanine, tyrosine and tryptophan.

The kinetics and mechanism of the oxidation of Glycine (Gly), Alanine (Ala), Tyrosine (Tyr), Tryptophan (Trp) and some di-(Gly-Gly, Ala-Ala, Gly-Ala, Gly-Trp, Trp-Gly, Gly-Tyr, Tyr-Gly), tri-(Gly-Gly-Gly, Ala-Gly-Gly) and tetrapeptides (Gly-Gly-Gly-Gly) mediated by sulfate (SO(4) (-)) and hydrogen phosphate (HPO(4) (-)) radicals was studied, employing the flash-photolysis technique. The substrates were found to react with sulfate radicals (SO(4) (-), produced by photolysis of the S(2)O(8)(2-)) faster than with hydrogen phosphate radicals (HPO(4) (-), generated by photolysis of P(2)O(8)(4-) at pH = 7.1). The reactions of the zwitterions of the aliphatic amino acids and peptides with SO(4) (-) radicals take place by electron transfer from the carboxylate moiety to the inorganic radical, whereas those of the HPO(4) (-) proceed by H-abstraction from the alpha carbon atom. The phenoxyl radical of Tyr-Gly and Gly-Tyr are formed as intermediate species of the oxidation of these peptides by the inorganic radicals. The radical cations of Gly-Trp and Trp-Gly (at pH = 4.2) and their corresponding deprotonated forms (at pH = 7) were detected as intermediates species of the oxidation of these peptides with SO(4) (-) and HPO(4) (-). Reaction mechanisms which account for the observed intermediates are proposed.     

Evaluation of DEPMPO as a spin trapping agent in biological systems

Cellular toxicity, pharmacokinetics, and the in vitro and in vivo stability of the SO3*- spin adduct of the spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide (DEPMPO), was investigated, and the results were compared with those of the widely used spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Similar to DMPO, DEPMPO was quickly taken up (<15 min) after intraperitoneal injection, and distributed evenly in the liver, heart, and blood of the mice. In the presence of ascorbate the in vitro stability of the adduct DEPMPO/SO3*- was 7 times better than DMPO/SO3*-. Under in vivo conditions, the spin adduct DEPMPO/SO3*- was 2-4 times more stable than DMPO/ SO3*-, depending on the route of administration of the adducts. Using a low frequency EPR spectrometer, we were able to observe the spin trapped SO3*- radical both with DMPO and DEPMPO directly in the intact mouse. DEPMPO had a detectable spin adduct signal at a concentration as low as 1 mM, as compared to 5 mM for DMPO. We conclude that DEPMPO is potentially a good candidate for trapping radicals in functioning biological systems, and represents an improvement over the commonly used trap DMPO.     

Study on the polarographic catalytic wave of vitamin P in the presence of persulfate and its application

The polarographic catalytic wave of vitamin P in the presence of persulfate was studied by linear potential scan polarography and cyclic voltammetry. Vitamin P yielded a single reduction wave in acidic aqueous solution, which was ascribed to a 2e(-), 2H(+) reduction of the carbonyl group in the C-4 position. Actually, the carbonyl group C=O first underwent a 1e(-), 1H(+) reduction to form a neutral free radical, and the further 1e(-), 1H(+) reduction of the free radical was simultaneous with its following chemical reactions. When S(2)O(2-)(8) was present, the free radical of vitamin P was oxidized by both S(2)O(2-)(8) and its reduction intermediate, the sulfate radical anion SO(*-)(4), to regenerate the original, which resulted in the production of a polarographic catalytic wave of vitamin P. Based on this catalytic wave, a novel method for the determination of vitamin P was proposed. In 0.02 M tartaric acid-sodium tartrate (pH 3.3) buffer containing 5.0 x 10(-3) M K(2)S(2)O(8), the peak potential of the catalytic wave was -1.42 V (vs SCE) and the peak current was rectilinear to the vitamin P concentration in the range of 8.0 x 10(-9)-1.0 x 10(-6) M (r = 0.9994, n = 13). The catalytic wave of 2.0 x 10(-7) M vitamin P enhanced the polarographic current 70 times compared with the corresponding reduction wave. The detection limit was 2.0 x 10(-9) M, and the relative standard deviation at the 2.0 x 10(-7) M level was 0.7% (n = 15). The proposed method was used for the determination of vitamin P content in the pharmaceutical preparation of tablets and the medicinal plant Sophora japonica L. without previous separation.     

Intramolecular rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen content

Variation of tissue oxygen content is thought to be a possible factor in determining the structural diversity of hydroperoxy fatty acids. In the present study, we evaluated the structural diversity of intermediate carbon-centered radicals at lower oxygen content. When the buffered solution (pH 7.4) containing 1.0 mM alpha-linolenic acid, 1.0 muM soybean 15-lipoxygenase, and 1.0 mM nitroxyl radical [3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmDeltaP)], which selectively traps carbon-centered radicals, was incubated in a sealed vial, the generation of linolenate hydroperoxide was completed within 1 min. In the subsequent reaction at lower oxygen content, the production of the [LnA-H+O(2)].-CmDeltaP adduct was ascertained by liquid chromatography tandem mass spectrometry with precursor ion scanning. Furthermore, HPLC analysis with photodiode array detection showed that the adduct exhibits an absorption maximum at 278 nm, indicating a conjugated triene moiety. On the basis of these facts, the structure of the adduct was speculated to be C(2)H(5)-CH(CmDeltaP)-CH = CH-CH = CH-CH = CH-CH(OOH) -C(7)H(14)-COOH. We proposed a possible reaction pathway as follows: a linolenate 9-peroxyl radical generated in the lipoxygenase reaction might be converted into C(2)H(5)-.CH-CH = CH-CH = CH-CH = CH-CH(OOH) -C(7)H(14)-COOH through an intramolecular rearrangement. This intermediate radical may give rise to hydroperoxy fatty acids with structural diversity.