Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

Different suicide inactivation processes for the peroxidase and cyclooxygenase activities of prostaglandin endoperoxide H synthase-1.

Prostaglandin endoperoxide H synthases (PGHSs)-1 and -2 have a cyclooxygenase (COX) activity involved in forming prostaglandin G2 (PGG2) from arachidonic acid and an associated peroxidase (POX) activity that reduces PGG2 to PGH2. Suicide inactivation processes are observed for both POX and COX reactions. Here we report COX reaction conditions for PGHS-1 under which complete COX inactivation occurs but with > or = 60% retention of POX activity. The rates of POX inactivation were compared for native oPGHS-1 versus Y385F oPGHS-1, a mutant that cannot form the Tyr385 radical of COX Intermediate II; the rates were the same for both native and Y385F oPGHS-1. Our data indicate that a COX Intermediate II/acyl or product complex is the precursor in COX inactivation. However, another species, probably an Intermediate II-like species but with a radical centered on a tyrosine other than Tyr385, is the immediate precursor for POX inactivation.     

Free radical generation during delta-aminolevulinic acid autoxidation: induction by hemoglobin and connections with porphyrinpathies

delta-Aminolevulinic acid (ALA), a heme precursor accumulated in acute intermittent porphyria and saturnism, undergoes autoxidation leading to ammonium ion and probably the corresponding alpha-ketoaldehyde. This reaction is accelerated by addition of oxyhemoglobin (oxyHb) and other iron complexes. OxyHb is concomitantly oxidized to metHb; the apparent second-order rate constant of oxyHb/ALA coupled oxidation is ca. 10 M-1 min-1.1H NMR and uv spectral studies suggest that ALA undergoes enolization before consuming the dissolved oxygen. Spin-trapping experiments demonstrate formation of both the hydroxyl radical and a substrate-derived carbon-centered radical during ALA oxidation. Generation of active oxygen species by ALA might be related to the neuropathy associated to some acquired and inherited porphyrinpathies.     

Effect of polar solvents on beta-carotene radical precursor

Beta-carotene forms radicals in chloroform upon photo-excitation (i) in the femtosecond time-scale by direct electron ejection into chloroform and (ii) in the microsecond time-scale by secondary reactions with chloroform radicals formed in the faster reactions. The precursor for beta-carotene radical cation decays in a second-order reaction in the mixed solvents, with a rate decreasing for increasing dielectric constant of cosolvent (acetic acid < ethanol < acetonitrile approximately methanol). The precursor is assigned as an ion pair from which the beta-carotene radical cation is formed in neat chloroform, but in more polar solvents it reacts at least partly through disproportionation in a bimolecular reaction promoted by the presence of ions. The stabilization of the radical precursor by increased solvent polarity, allowing for deactivation of the precursor by an alternative reaction channel, is discussed in relation to the balance of pro- and antioxidative properties of beta-carotene at lipid/water interfaces.     

Inactivation of the 2-oxo acid dehydrogenase complexes upon generation of intrinsic radical species.

Self-regulation of the 2-oxo acid dehydrogenase complexes during catalysis was studied. Radical species as side products of catalysis were detected by spin trapping, lucigenin fluorescence and ferricytochrome c reduction. Studies of the complexes after converting the bound lipoate or FAD cofactors to nonfunctional derivatives indicated that radicals are generated via FAD. In the presence of oxygen, the 2-oxo acid, CoA-dependent production of the superoxide anion radical was detected. In the absence of oxygen, a protein-bound radical concluded to be the thiyl radical of the complex-bound dihydrolipoate was trapped by alpha-phenyl-N-tert-butylnitrone. Another, carbon-centered, radical was trapped in anaerobic reaction of the complex with 2-oxoglutarate and CoA by 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Generation of radical species was accompanied by the enzyme inactivation. A superoxide scavenger, superoxide dismutase, did not protect the enzyme. However, a thiyl radical scavenger, thioredoxin, prevented the inactivation. It was concluded that the thiyl radical of the complex-bound dihydrolipoate induces the inactivation by 1e- oxidation of the 2-oxo acid dehydrogenase catalytic intermediate. A product of this oxidation, the DMPO-trapped radical fragment of the 2-oxo acid substrate, inactivates the first component of the complex. The inactivation prevents transformation of the 2-oxo acids in the absence of terminal substrate, NAD+. The self-regulation is modulated by thioredoxin which alleviates the adverse effect of the dihydrolipoate intermediate, thus stimulating production of reactive oxygen species by the complexes. The data point to a dual pro-oxidant action of the complex-bound dihydrolipoate, propagated through the first and third component enzymes and controlled by thioredoxin and the (NAD+ + NADH) pool.     

EPR spin trapping of a radical intermediate in the urate oxidase reaction

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (Porcine Liver, Bacillus Fastidiosus, Candida Utilitis) was performed in the presence of 5-diethoxyphosphoryl-5-methyl-pyrroline-N-oxide (DEPMPO) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.     

EPR Spin Trapping of a Radical Intermediate in the Urate Oxidase Reaction

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (Porcine Liver, Bacillus Fastidiosus, Candida Utilitis) was performed in the presence of 5‐diethoxyphosphoryl‐5‐methyl‐pyrroline‐N‐oxide (DEPMPO) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.     

Intermediates in the reaction of substrate-free cytochrome P450cam with peroxy acetic acid

Freeze-quenched intermediates of substrate-free cytochrome 57Fe-P450(cam) in reaction with peroxy acetic acid as oxidizing agent have been characterized by EPR and Mossbauer spectroscopy. After 8 ms of reaction time the reaction mixture consists of approximately 90% of ferric low-spin iron with g-factors and hyperfine parameters of the starting material; the remaining approximately 10% are identified as a free radical (S' = 1/2) by its EPR and as an iron(IV) (S= 1) species by its Mossbauer signature. After 5 min of reaction time the intermediates have disappeared and the Mossbauer and EPR-spectra exhibit 100% of the starting material. We note that the spin-Hamiltonian analysis of the spectra of the 8 ms reactant clearly reveals that the two paramagnetic species, e.g. the ferryl (iron(IV)) species and the radical, are not exchanged coupled. This led to the conclusion that under the conditions used, peroxy acetic acid oxidized a tyrosine residue (probably Tyr-96) into a tyrosine radical (Tyr*-96), and the iron(III) center of substrate-free P450(cam) to iron(IV).     

Electron spin resonance studies of the reaction of hypochlorite with 5,5-dimethyl-1-pyrroline-N-oxide

The reaction of hypochlorous acid with the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was found to yield 5,5-dimethyl-2-pyrrolidone-N-oxyl (DMPOX). In addition to DMPOX, 5,5-dimethyl-2-hydroxypyrrolidine-N-oxyl (DMPO-OH) and an unidentified chlorine-containing radical species were also observed under neutral and near-neutral conditions. Through the use of [17O]HOCl and the hydroxyl radical scavengers ethanol and formate, it was established that DMPO-OH was derived from hydration of DMPO rather than the spin-trapping of hydroxyl radical. Furthermore, kinetic studies and the incorporation of 17O showed that DMPO-OH was readily oxidized to DMPOX and that this reaction was acid and base catalyzed. Under strongly alkaline conditions, DMPOX reversibly formed another species, presumably the enolate, that had a four-line ESR signal identical to that of DMPO-OH. Eventually, carbon-centered adducts appeared whose ESR signals were consistent with the formation of DMPO condensation products.     

Transient quinonimines and 1,4-benzothiazines of pheomelanogenesis: new pulse radiolytic and spectrophotometric evidence.

Biosynthetic and model in vitro studies have shown that pheomelanins, the distinctive pigments of red human hair, arise by oxidative cyclization of cysteinyldopas mainly 5-S-cysteinyldopa (1) via a critical o-quinonimine intermediate, which rearranges to unstable 1,4-benzothiazines. To get new evidence for these labile species, fast time resolution pulse radiolytic oxidation by dibromide radical anion of a suitable precursor, the dihydro-1,4-benzothiazine-3-carboxylic acid 7 was performed in comparison with that of 1. In the case of 7, dibromide radical anion oxidation leads over a few microseconds (k = 2.1 x 10(9) M(-1) s(-1)) to a phenoxyl radical (lambda(max) 330 nm, epsilon = 6300 M(-1) cm(-1)) which within tens of milliseconds gives rise with second-order kinetics (2k = 2.7 x 10(7) M(-1) s(-1)) to a species exhibiting an absorption maximum at 540 nm (epsilon = 2200 M(-1) cm(-1)). This was formulated as the o-quinonimine 3 arising from disproportionation of the initial radical. The quinonimine chromophore is converted over hundreds of milliseconds (k = 6.0 s(-1)) to a broad maximum at around 330 nm interpreted as due to a 1,4-benzothiazine or a mixture of 1,4-benzothiazines, which as expected are unstable and subsequently decay over a few seconds (k = 0.5 s(-1)). Interestingly, the quinonimine is observed as a labile intermediate also in the alternative reaction route examined, involving cyclization of the o-quinone (lambda(max) 390 nm, epsilon = 6900 M(-1) cm(-1)) arising by disproportionation (2k = 1.7 x 10(8) M(-1) s(-1)) of an o-semiquinone (lambda(max) 320 nm, epsilon = 4700 M(-1) cm(-1)) directly generated by dibromide radical anion oxidation of 1. Structural formulation of the 540 nm species as an o-quinonimine was further supported by rapid scanning diode array spectrophotometric monitoring of the ferricyanide oxidation of a series of model dihydrobenzothiazines.     

Interaction of tert-butyl hydroperoxide with hypochlorous acid. A spin trapping and chemiluminescence study

The formation of radical species during the reaction of ter-tbutyl hydroperoxide and hypochlorous acid has been investigated by spin trapping and chemiluminescence. A superposition of two signals appeared incubating tert-butyl hydroperoxide with hypochlorous acid in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The first signal (aN = 1.537 mT, aH beta = 0.148 mT) was an oxidation product of POBN caused by the action of hypochlorous acid. The second spin adduct (aN = 1.484 mT, aH beta = 0.233 mT) was derived from a radical species that was formed in the result of reaction of tert-butyl hydroperoxide with hypochlorous acid. Similarly, a superposition of two signals was also obtained using the spin trap N-tert-butyl-alpha-phenylnitrone (PBN). tert-Butyl hydroperoxide was also treated with Fe2+ or Ce4+ in the presence of POBN. Using Fe2+ a spin adduct with a N = 1.633 mT and aH beta = 0.276 mT was observed. The major spin adduct formed with Ce4+ was characterised by a N = 1.480 mT and aH beta = 0.233 mT. The reaction of tert-butyl hydroperoxide with hypochlorous acid was accompanied by a light emission, that time profile and intensity were identical to those emission using Ce4+. The addition of Fe2+ to tert-butyl hydroperoxide yielded a much smaller chemiluminescence. Thus, tert-butyl hydroperoxide yielded in its reaction with hypochlorous acid or Ce4+ the same spin adduct and the same luminescence profile. Because Ce4+ is known to oxidize organic hydroperoxides to peroxyl radical species, it can be concluded that a similar reaction takes place in the case of hypochlorous acid.     

Detection of Myoglobin-Derived Radicals On Reaction of Metmyoglobin With Hydrogen Peroxide and Other Peroxidic Compounds

The reaction of metmyoglobin with equimolar concentrations of hydrogen peroxide has been studied using both electron spin resonance (e.s.r.) and optical spectroscopy. Using the former technique a strong anisotropic e.s.r. signal is observed, in the presence of the spin trap DMPO, which decays relatively rapidly. This previously unobserved signal, which is also observed on reaction of metmyoglobin with a number of other powerful oxidants (peracetic acid, 3-chloroperoxybenzoic acid, monoperoxyphthalic acid, iodosyl benzene, ¹BuOOH and cumene hydroperoxide) is assigned to a slowly-tumbling, metmyoglobin-derived, spin adduct. The parameters of this signal (a_N 1. 45, a_H 0.83 mT) are consistent with the trapped radical having a heteroatom centre; this is believed to be oxygen. The concentration of this species is not affected by compounds such as 2-deoxyribose, mannitol and phenylalanine which are all efficient hydroxyl radical scavengers, demonstrating that the formation of this radical is not due to reaction of “free” HO· generated by breakdown of H₂O₂, by released iron ions. The concentration of this species is however decreased by desferal, ascorbate, Trolox C, salicylate and, to a lesser extent, linoleic acid; with the first three of these compounds further substrate-derived radicals are also observed. Examination of similar reaction systems (though in the absence of DMPO) by optical spectroscopy shows that the myoglobin (IV) species is formed and that this species behaves in a somewhat different manner with these added compounds. These results suggest that the radical trapped in the e.s.r. experiments is a myoglobin-derived species, probably a tyrosine peroxyl radical, arising from oxidative damage to the globin moiety.

The diminution of both the e.s.r. signal of the spin adduct and the optical absorption of the myoglobin (IV) species in the presence of linoleic acid suggests that these myoglobin-derived species can initiate oxidative damage but that this process can be ameliorated by the presence of a number of water-soluble compounds such as ascorbate, Trolox C, desferal and salicylate.     

Ceriporic acid B, an extracellular metabolite of Ceriporiopsis subvermispora, suppresses the depolymerization of cellulose by the Fenton reaction

The white rot fungus, Ceriporiopsis subvermispora, is able to degrade lignin in wood without intensive damage to cellulose. Since lignin biodegradation by white rot fungi proceeds by radical reactions, accompanied by the production of a large amount of Fe3+-reductant phenols and reductive radical species in the presence of iron ions, molecular oxygen, and H2O2, C. subvermispora has been proposed to possess a biological system which suppresses the production of a cellulolytic active oxygen species, *OH, by the Fenton reaction. In the present paper, we demonstrate that 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B), an extracellular metabolite of C. subvermispora, strongly inhibited *OH production and the depolymerization of cellulose by the Fenton reaction in the presence of iron ions, cellulose, H2O2, and a reductant for Fe3+, hydroquinone (HQ), at the physiological pH of the fungus.     

Evidence for a free radical chain mechanism in the reaction between peroxidase and indole-3-acetic acid at neutral pH

The oxidation of indole-3-acetic acid (IAA) catalyzed by horseradish peroxidase (HRP) in the absence of added H2O2 was studied at pH 7.4 using spectral and kinetic approaches. Upon addition of a hundred-fold excess of IAA to HRP the native enzyme was rapidly transformed to compound II (HRP-II). HRP-II was the predominant catalytic enzyme species during the steady state. No compound III was observed. HRP-II was slowly transformed to the stable inactive verdohemo-protein, P-670. A precursor of P-670, so-called P-940 was not detected. After the cessation of IAA oxidation there was neither oxygen consumption nor P-670 formation; the remaining HRP-II was spontaneously reduced to native enzyme. Single exponential kinetics were observed in the steady state for IAA oxidation, oxygen consumption and P-670 formation yielding identical first order rate constants of about 6 . 10(4) s(-1). A comparison of the rate of IAA oxidation by HRP-II in the steady state and in the transient state indicated that more than 1 3 of the IAA was oxidized non-enzymatically during the steady state, confirming that a free radical chain reaction is involved in the peroxidase-catalyzed oxidation of IAA. IAA oxidation stopped before IAA was completely consumed, which cannot be ascribed to enzyme inactivation because 30-50% of the enzyme was still active after the end of the reaction. Instead, incomplete IAA oxidation is explained in terms of termination of the free radical chain reaction. Bimolecular rate constants of IAA oxidation by HRP-I and HRP-II determined under transient state conditions were (2.2 +/- 0.1) x 10(3) M(-1) s(-1) and (2.3 +/- 0.2) x 10(2) M(-1) s(-1).     

Oxidation of vitamin E during iron-catalyzed lipid peroxidation: evidence for electron-transfer reactions of the tocopheroxyl radical.

Incubation of phosphatidylcholine liposomes containing the biological antioxidant alpha-tocopherol (alpha-TH) with xanthine, xanthine oxidase, and FeCl2 caused alpha-TH oxidation to alpha-tocopherol quinone (alpha-TQ) and 8a-hydroperoxytocopherone (2). In addition, 4a,5-epoxy-8a-hydroperoxytocopherone (3), 7,8-epoxy-8a-hydroperoxytocopherone (4), and their respective hydrolysis products 2,3-epoxy-alpha-tocopherol quinone (6) and 5,6-epoxy-alpha-tocopherol quinone (7) also were formed. alpha-TQ was the major product at less than 20% alpha-TH oxidation, whereas epoxides were the predominant products when alpha-TH was more extensively oxidized. 8a-(Alkyldioxy)tocopherones 1, which are formed when peroxyl radicals oxidize alpha-TH in other systems and which are precursors to alpha-TQ, were not found. 8a-Hydroxytocopherone (5), rather than 8a-(alkyldioxy)tocopherones 1, appeared to be the precursor to alpha-TQ. Approximately 30% of the alpha-TH consumed was regenerated by treatment of samples with ascorbic acid or nordehydroguaiaretic acid (NDGA) at pH 3, but not at pH 7. The stability of the ascorbic acid- and NDGA-reducible species and pH dependence for regeneration matched those of 8a-hydroxytocopherone (5) and contrasted with the properties of the tocopheroxyl radical (alpha-T.). Incubation of liposomes containing alpha-TH with the diphenylpicrylhydrazyl (DPPH) radical, which oxidizes alpha-TH to alpha-T. in high yield, formed an ascorbic acid-reducible species with properties identical to those of compound 5. The results indicate that phospholipid peroxyl radicals oxidize alpha-T. to epoxides, 8a-hydroperoxytocopherone (2), and the tocopherone cation (alpha-T+), which hydrolyzes to 5, the immediate precursor to alpha-TQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of generation of oxygen radicals and reductive mobilization of ferritin iron by lipoamide dehydrogenase.

The oxidase reaction of lipoamide dehydrogenase with NADH generates superoxide radicals and hydrogen peroxide under aerobic conditions. ESR spin trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to characterize the oxygen radical species generated by lipoamide dehydrogenase and the mechanism of their generation. During the oxidase reaction of lipoamide dehydrogenase, DMPO-OOH and DMPO-OH signals were observed. The DMPO-OOH signal disappeared on addition of superoxide dismutase. These results demonstrate that the DMPO-OOH adduct was produced from the superoxide radical generated by lipoamide dehydrogenase. In the presence of dimethyl sulfoxide, a DMPO-CH3 signal appeared at the expense of the DMPO-OH signal, indicating that the DMPO-OH adduct was produced directly from the hydroxyl radical rather than by decomposition of the DMPO-OOH adduct. The DMPO-OH signal decreased on addition of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, indicating that the hydroxyl radical was generated via the metal-catalyzed Haber-Weiss reaction from the superoxide radical and hydrogen peroxide. Addition of ferritin to the NADH-lipoamide dehydrogenase system resulted in a decrease of the DMPO-OOH signal, indicating that the superoxide radical interacted with ferritin iron.     

Isolation and identification of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts formed by the decomposition of the hydroperoxides of linoleic acid, linolenic acid, and arachidonic acid by soybean lipoxygenase.

alpha-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, which are formed in the reactions of soybean lipoxygenase with linoleic acid, arachidonic acid, and linolenic acid, were isolated using HPLC-ESR spectroscopy. Both linoleic acid and arachidonic acid gave one radical adduct, whereas in the case of linolenic acid, two radical adducts were isolated. These radical adducts all showed virtually identical uv spectra with lambda max at 292 and 220 nm in hexane. The absence of absorbance with lambda max at 234 nm indicates that a conjugated diene structure is not contained in these radical adducts. The mass spectra of the radical adducts formed from linoleic and arachidonic acids were identical and contained a molecular ion of m/z 264, consistent with the trapping of the pentyl radical by 4-POBN. Indeed, authentic 4-POBN pentyl radical adduct obtained from the reaction between pentylhydrazine and 4-POBN gave the same mass spectrum as the product obtained from the reaction of linoleic acid and arachidonic acid with 4-POBN. The two 4-POBN radical adducts formed in the linolenic acid reaction were shown by mass spectrometry to be isomers of pentenyl radicals. The 4-POBN-pentyl radical adduct was also detected in the reaction mixture of 13-hydroperoxy-linoleic acid, soybean lipoxygenase, and 4-POBN, indicating that the pentyl radical and pentenyl radical are formed by the decomposition of the hydroperoxides.     

A free radical mechanism of prostaglandin synthase-dependent aminopyrine demethylation

The mechanism of prostaglandin synthase-dependent N-dealkylation has been investigated using an enzyme preparation derived from ram seminal vesicles. Incubation of an N-alkyl substrate, aminopyrine, with enzyme and arachidonic acid, 15-hydroperoxyarachidonic acid, or tert-butyl hydroperoxide resulted in the formation of the transient aminopyrine free radical species. Formation of this radical species, which was detected by electron paramagnetic resonance spectroscopy and/or absorbance at 580 nm, was maximal approximately 30 s following initiation of the reaction and declined thereafter. Free radical formation corresponded closely with formaldehyde formation in this system, in terms of dependence upon substrate and cofactor concentration, as well as in terms of time course. Both aminopyrine free radical and formaldehyde formation were inhibited by indomethacin and flufenamic acid, inhibitors of prostaglandin synthase. The results suggest that the aminopyrine free radical is an intermediate in the prostaglandin synthase-dependent aminopyrine N-demethylase pathway. The aminopyrine free radical electron paramagnetic resonance spectrum revealed that this species is a one-electron oxidized cation radical of the parent compound. A reaction mechanism has been proposed in which aminopyrine undergoes two sequential one-electron oxidations to an iminium cation, which is then hydrolyzed to the demethylated amine and formaldehyde. Accordingly, the oxygen atom of the aldehyde product is derived from neither molecular nor hydroperoxide oxygen, but from water.     

Formation Mechanism of the Free Radical Product and Its Precursor by the Reaction of Dehydro-L-ascorbic Acid with Amino Acid

During the formation of radical A (2) and its precursor (tris(2-deoxy-2-L-ascorbyl)amine, 1) by the reaction of dehydroascorbic acid (DHA) with amino acid, ascorbic acid (AsA) and the reduced red pigment (3) were newly identified, in addition to scorbamic acid (SCA) and the red pigment (4), as intermediate products. The addition of AsA to the DHA-amino acid reaction, as well as to the DHA-SCA reaction, greatly increased the formation of 3 and 1. The reaction of AsA with 4 gave rapidly 3, followed by the gradual production of 1. From these results, a reaction pathway is proposed that 3 formed by the reduction of 4 with AsA is a key intermediate and its condensation with DHA followed by reduction with AsA might produce 2 and 1.     

The free radical formed during the hydroperoxide-mediated deactivation of ram seminal vesicles is hemoprotein-derived

Prostaglandin synthase is a multi-enzyme complex which catalyzes the oxygenation of arachidonic acid to the various prostaglandins. During the oxygenation, the enzyme is self-deactivated and, on the basis of ESR data, it has been proposed to form a self-destructive free radical. The free radical was suggested to form from the oxygen lost from prostaglandin G2 during its reduction to prostaglandin H2, and the destructive species was therefore thought to be an oxygen-centered free radical, tentatively identified as the hydroxy radical. We have reinvestigated this ESR signal (g = 2.005) and have concluded, with the aid of the known ESR parameters for the hydroxy and other oxygen-centered free radicals, that the free radical formed during the oxygenation is neither a hydroxy nor any known oxygen-centred radical. Prostaglandin synthase is thought to be a hemoprotein, so this unknown ESR signal was compared with the previously observed free radical formed by the reaction of H2O2 with methemoglobin. This comparison indicates that the free radical formed by the reaction of prostaglandin G2 with ram seminal vesicles is hemoprotein-derived and may be formed by the oxidation of an amino acid(s) located near the iron of the heme.