Mechanism of horseradish peroxidase-catalyzed heme oxidation and polymerization (beta-hematin formation)

Horseradish peroxidase (HRP) catalyzes the polymerization of free heme (beta-hematin formation) through its oxidation. Heme when added to HRP compound II (FeIV=O) causes spectral shift from 417 nm (Compound II) to 402 nm (native, FeIII) indicating that heme may be oxidized via one-electron transfer. Direct evidence for one-electron oxidation of heme by HRP intermediates is provided by the appearance of an E.s.r signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-heme radical adduct (a1H=14.75 G, a2H=4.0 G) in E.s.r studies. Heme-polymerization by HRP is inhibited by spin trap indicating that one-electron oxidation product of heme ultimately leads to the formation of heme-polymer. HRP, when incubated with diethyl pyrocarbonate (DEPC), a histidine specific reagent, shows concentration dependent loss of heme-polymerization indicating the role of histidine residues in the process. We suggest that HRP catalyzes the formation of heme-polymer through one-electron oxidation of free heme.     

Characteristics of chemiluminescence observed in the horseradish peroxidase-hydrogen peroxide-tyrosine system.

Electrolysis or horseradish peroxidase (HRP)-catalyzed oxidation of tyrosine and bityrosine in aqueous solution at pH 7.4 resulted in light emission in the visible region. Electrolysis of tyrosine emitted light which peaked at 490 nm and was almost completely quenched by superoxide dismutase (SOD), while emission by bityrosine peaked at 530 nm. In the HRP-H(2)O(2)-tyrosine system the oxidation-reduction of tyrosine emitted light with two prominent peaks, 490 and 530 nm, and was not quenched by SOD. The phenoxyl neutral radical of the tyrosine in HRP-H(2)O(2)-tyrosine system was detected by electron spin resonance (ESR) spectrometry using tert-nitrosobutane as a spin trap; the spin adduct was found to adhere to the HRP molecule during the enzymatic reaction. Further, bityrosine was detected in the HRP-H(2)O(2)-tyrosine reaction system. Changes in absorption spectra of HRP and chemiluminescence intensities during HRP-catalyzed oxidation of tyrosine suggest that for photon emission compound III is a candidate superoxide donor to the phenoxyl cation radical of tyrosine on the enzyme molecule. The luminescence observed in this study might be originated from at least two exciplexes involved with the tyrosine cation radical (Tyr(*+)) and the bityrosine cation radical (BT(*+))     

Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide

It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle. Chlorite mediates the two-electron oxidation of ferric HRP to compound I (k(1)) thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of both compound I (k(2)) and compound II (k(3)) forming chlorine dioxide. The strong pH-dependence of all three reactions clearly suggests that chlorous acid is the reactive species. Typical apparent bimolecular rate constants at pH 5.6 are 1.4 x 10(5)M(-1)s(-1) (k(1)), 2.25 x 10(5)M(-1)s(-1) (k(2)), and 2.4 x 10(4)M(-1)s(-1) (k(3)), respectively. Moreover, the reaction products hypochlorous acid and chlorine dioxide, which are known to induce heme bleaching and amino acid modification upon longer incubation times, also mediate the oxidation of ferric HRP to compound I (2.4 x 10(7)M(-1)s(-1) and 2.7 x 10(4)M(-1)s(-1), respectively, pH 5.6) but do not react with compounds I and II. A reaction scheme is presented and discussed from both a mechanistic and thermodynamic point of view. It helps to explain the origin of contradictory data so far found in the literature on this topic.     

The metal-catalyzed oxidation of methionine in peptides by Fenton systems involves two consecutive one-electron oxidation processes.

The one-electron oxidation of methionine (Met) plays an important role in the redox reactions of Met in peptides and proteins under conditions of oxidative stress, e.g., during the metal-catalyzed oxidation of beta-amyloid peptide (beta A). However, little information is available with regard to mechanisms and product formation during the metal-catalyzed oxidation of Met. Here, we demonstrate that two-electron oxidation of Met in Fenton reactions, carried out aerobically by [Fe(II)(EDTA)](2-) and H(2)O(2) (EDTA = ethylenediaminetetra acetate) is the consequence of two consecutive one-electron transfer reactions carried out by either free or complexed hydroxyl radicals, followed by the reaction of an intermediary sulfur-nitrogen bonded radical cation (sulfuranyl radical) with O(2). The model peptide Met-Met represents an ideal substrate for these investigations as its one-electron oxidation, followed by reaction with molecular oxygen, produces unique intermediates, azasulfonium diastereomers, which can be chemically isolated before hydrolysis to sulfoxide occurs.     

Spectroscopic properties and reactivity of free radical forms of A2E

A pyridinium bisretinoid (A2E) is the only identified blue-absorbing chromophore of retinal lipofuscin that has been linked to its aerobic photoreactivity and phototoxicity. Pulse radiolysis has been used to study both the one-electron oxidation and the one-electron reduction of A2E in aqueous micellar solutions. The reduction to the semireduced A2E (lambda(max) broad and between 500 and 540 nm) was achieved with formate radicals and the subsequent decay of A2E* was slow (over hundreds of milliseconds) via complex kinetics. The long lifetime of the A2E* should facilitate its reactions with other biomolecules. For example, with oxygen, the A2E* produced the superoxide radical anion with a rate constant of 3 x 10(8) M(-1) s(-1). The A2E was also reduced by the NAD radical, the corresponding rate constant being 2.3 x 10(8) M(-1) s(-1). Other experiments showed that the one-electron reduction potential of A2E lies in the range -640 to -940 mV. The semioxidized form of A2E (lambda(max) 590 nm) was formed via oxidation with the Br2*- radical and had a much shorter lifetime than the semireduced form. With strongly oxidizing peroxyl radicals (CCl3O2*) our kinetic data suggest the formation of a radical adduct followed by dissociation to the semioxidized A2E. With milder oxidizing peroxyl radicals such as that from methanol, our results were inconclusive. In benzene we observed an efficient oxidation of zeaxanthin to its radical cation by the A2E radical cation; this may be relevant to a detrimental effect of A2E in vision.     

One- and two-electron reduction of ubiquinone homologs by NADH- dehydrogenase preparations from the mitochondrial respiratory chain

The mechanism of ubiquinone homologs reduction by different preparations of mitochondrial NADH dehydrogenase: complex I within submitochondrial particles, isolated NADH-ubiquinone oxidoreductase and soluble low molecular weight NADH dehydrogenase, has been investigated. It has been shown that NADH oxidation via the rotenone-insensitive reaction is associated with one-electron reduction of low molecular weight ubiquinone homologs (Q0, Q1, Q2) to semiquinone with subsequent fast oxidation of the latter by atmospheric oxygen to form a superoxide radical. The two-electron ubiquinone reduction to quinol in the rotenone-sensitive reaction is unaccompanied by the semiquinone release from the enzyme active center into the surrounding solution.     

Characterization of one- and two-electron oxidations of glutathione coupled with lactoperoxidase and thyroid peroxidase reactions

Glutathione (GSH) was oxidized to GSSG in the presence of H2O2, tyrosine, and peroxidase. During the GSH oxidation catalyzed by lactoperoxidase, O2 was consumed and the formation of glutathione free radical was confirmed by ESR of its 5,5'-dimethyl-1-pyrroline-N-oxide adduct. When lactoperoxidase was replaced by thyroid peroxidase in the reaction system, the consumption of O2 and the formation of the free radical became negligibly small. These results led us to conclude that, in the presence of H2O2 and tyrosine, lactoperoxidase and thyroid peroxidase caused the one-electron and two-electron oxidations of GSH, respectively. It was assumed that GSH is oxidized by primary oxidation products of tyrosine, which are phenoxyl free radicals in lactoperoxidase reactions and phenoxyl cations in thyroid peroxidase reactions. When tyrosine was replaced by diiodotyrosine or 2,6-dichlorophenol, the difference in the mechanism between lactoperoxidase and thyroid peroxidase disappeared and both caused the one-electron oxidation of GSH. Iodides also served as an effective mediator of GSH oxidation coupled with the peroxidase reactions. In this case the two peroxidases both caused the two-electron oxidation of GSH.     

Mechanistic study of the oxidation of caffeic acid by digital simulation of cyclic voltammograms

The oxidation mechanism of caffeic acid (CAF) has been studied by means of cyclic voltammetry with the plastic formed carbon or glassy carbon electrode. CAF gives a well-developed two-electron reversible wave in acidic media, whereas it shows an irreversible behavior, i.e., a decrease of the rereduction peak, in less acidic media, suggesting that the oxidation of CAF follows an irreversible chemical reaction(s). Digital simulation analyses based on different oxidation mechanisms have been performed for the voltammograms obtained with the GC electrode in 1:1 (v/v) water:ethanol solutions. The results clearly show that the seeming two-electron oxidation of CAF occurs stepwise via one-electron processes, each of which follows an irreversible chemical reaction. It has also been suggested that the semiquinone radical as an intermediate of the one-electron oxidation should play an important role in the oxidation reaction. Evaluations of the rate constants for the chemical reactions have further suggested that the chemical reactions are dimerization reactions.     

Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity

The free radical scavenging properties and possible antioxidant activity of folic acid are reported. Pulse radiolysis technique is employed to study the one-electron oxidation of folic acid in homogeneous aqueous solution. The radicals used for this study are CCl₃O₂^•, N₃^•, SO₄^•−, Br₂^•−, √OH, and O^•−. All these radicals react with folic acid under ambient condition at an almost diffusion-controlled rate producing two types of transients. The first transient absorption maximum is around 430 nm, which decays, and a simultaneous growth at around 390 nm is observed. Considering the chemical structure of folic acid, the absorption maximum at 430 nm has been assigned to a phenoxyl radical. The latter one is proposed to be a delocalized molecular radical. A permanent product has been observed in the oxidation of folic acid with CCl₃O₂^• and N₃^• radicals, with a broad absorption band around 370–400 nm. The bimolecular rate constants for all the radical-induced oxidation reactions of folic acid have been measured. Folic acid is seen to scavenge these radicals very efficiently. In the reaction of thiyl radicals with folic acid, it has been observed that folic acid can not only scavenge thiyl radicals but can also repair these thiols at physiological pH. While carrying out the lipid peroxidation study, in spite of the fact that folic acid is considerably soluble in water, we observed a significant inhibition property in microsomal lipid peroxidation. A suitable mechanism for oxidation of folic acid and repair of thiyl radicals by folic acid has been proposed.     

Reaction of ferrous lactoperoxidase with hydrogen peroxide and dioxygen: an anaerobic stopped-flow study

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions including milk, tears, and saliva and has physiological significance in antimicrobial defense which involves (pseudo-)halide oxidation. LPO compound III (a ferrous-dioxygen complex) is known to be formed rapidly by an excess of hydrogen peroxide and could participate in the observed catalase-like activity of LPO. The present anaerobic stopped-flow kinetic analysis was performed in order to elucidate the catalytic mechanism of LPO and the kinetics of compound III formation by probing the reactivity of ferrous LPO with hydrogen peroxide and molecular oxygen. It is shown that ferrous LPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl LPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (7.2+/-0.3) x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C. The H2O2-mediated conversion of compound II to compound III follows also second-order kinetics (220 M(-1) s(-1) at pH 7.0 and 25 degrees C). Alternatively, compound III is also formed by dioxygen binding to ferrous LPO at an apparent bimolecular rate constant of (1.8+/-0.2) x 10(5) M(-1) s(-1). Dioxygen binding is reversible and at pH 7.0 the dissociation constant (K(D)) of the oxyferrous form is 6 microM. The rate constant of dioxygen dissociation from compound III is higher than conversion of compound III to ferric LPO, which is not affected by the oxygen concentration and follows a biphasic kinetics. A reaction cycle including the redox intermediates compound II, compound III, and ferrous LPO is proposed, which explains the observed (pseudo-)catalase activity of LPO in the absence of one-electron donors. The relevance of these findings in LPO catalysis is discussed.     

DNA and 2'-deoxyguanosine damage in the horseradish-peroxidase-catalyzed autoxidation of aldehydes: the search for the oxidizing species

The horseradish-peroxidase(HRP)-catalyzed aerobic oxidation of aldehydes, in particular isobutanal, was used for the oxidative damage of DNA. In isolated calf-thymus DNA, the enzymatic oxidation of isobutanal led to 7,8-dihydro-8-oxoguanine (8-oxoGua) in up to 1.3% yield and appreciable single-strand breaks in supercoiled pBR 322 DNA. For the nucleoside dG, significant amounts of the guanidine-releasing products oxazolone and oxoimidazolidine have been detected, but 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) was not obtained. Only enolizable aldehydes are effective, molecular oxygen is essential, and radical scavengers inhibit efficiently the oxidation. Comparative experiments with 3,3,4,4-tetramethyl-1,2-dioxetane (TMD) revealed that triplet-excited acetone does not play a significant role in this enzymatic DNA oxidation. 2-Hydroperoxy-2-methylpropanal, an intermediate in the HRP-catalyzed aerobic oxidation of isobutanal, does not contribute directly in the observed dG conversion. However, the peroxyl radical derived from the 2-hydroperoxy-2-methylpropanal appears to be active as oxidant because model studies with a structurally related peroxyl radical, produced by HRP-catalyzed one-electron oxidation of 3-hydroperoxy-3-methyl-2-butanone, causes both dG conversion and DNA strand breaks, but to a moderate extent. The active oxidant, as established by control experiments, is the peroxyisobutyric acid, that is efficiently formed through the HRP-catalyzed autoxidation of isobutanal. Still more effective is the acylperoxyl radical, conveniently generated from the peracid by one-electron oxidation by HRP.     

Crystallographic, kinetic, and spectroscopic study of the first ligninolytic peroxidase presenting a catalytic tyrosine

Trametes cervina lignin peroxidase (LiP) is a unique enzyme lacking the catalytic tryptophan strictly conserved in all other LiPs and versatile peroxidases (more than 30 sequences available). Recombinant T. cervina LiP and site-directed variants were investigated by crystallographic, kinetic, and spectroscopic techniques. The crystal structure shows three substrate oxidation site candidates involving His-170, Asp-146, and Tyr-181. Steady-state kinetics for oxidation of veratryl alcohol (the typical LiP substrate) by variants at the above three residues reveals a crucial role of Tyr-181 in LiP activity. Moreover, assays with ferrocytochrome c show that its ability to oxidize large molecules (a requisite property for oxidation of the lignin polymer) originates in Tyr-181. This residue is also involved in the oxidation of 1,4-dimethoxybenzene, a reaction initiated by the one-electron abstraction with formation of substrate cation radical, as described for the well known Phanerochaete chrysosporium LiP. Detailed spectroscopic and kinetic investigations, including low temperature EPR, show that the porphyrin radical in the two-electron activated T. cervina LiP is unstable and rapidly receives one electron from Tyr-181, forming a catalytic protein radical, which is identified as an H-bonded neutral tyrosyl radical. The crystal structure reveals a partially exposed location of Tyr-181, compatible with its catalytic role, and several neighbor residues probably contributing to catalysis: (i) by enabling substrate recognition by aromatic interactions; (ii) by acting as proton acceptor/donor from Tyr-181 or H-bonding the radical form; and (iii) by providing the acidic environment that would facilitate oxidation. This is the first structure-function study of the only ligninolytic peroxidase described to date that has a catalytic tyrosine.     

Characterization of the transient species generated by the photoexcitation of C-phycocyanin from Spirulina platensis: a laser photolysis and pulse radiolysis study

Nanosecond laser flash photolysis and pulse radiolysis were used to generate and characterize the triplet state and cation radical of C-phycocyanin (C-PC) from Spirulina platensis. The transient absorption spectra of C-PC were measured from direct excitation and acetone sensitization in aqueous solution at room temperature by KrF (248 nm) laser flash photolysis. Laser-induced transient species have been characterized by the method of acetone sensitization and one-electron oxidation. In nitrous oxide-saturated phosphate buffer saline (pH = 7.0) of C-PC, the produced intermediates are assigned to the excited triplet state and the radical cation. Using acetone as photosensitizer, the C-PC excited triplet states produced via triplet-triplet energy transfer and the C-PC radical cation from electron transfer reaction were further confirmed. Furthermore, the corresponding kinetic parameters were determined. To our knowledge, the transient absorption spectra of C-PC have been reported for the first time.     

Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer

Glutathione reductase has been found to catalyze an NAD(P)H-dependent electron transfer to 2,4,6-trinitrobenzenesulfonate (TNBS). In the presence of oxygen TNBS is not consumed in the reaction, but is rapidly reoxidized with concomitant production of hydrogen peroxide. Cytochrome c can replace oxygen as the final electron acceptor, indicating that a one-electron transfer takes place. The rate is slightly higher in the absence than in the presence of oxygen, ruling out superoxide anion as an obligatory intermediate in cytochrome c reduction. In the absence of oxygen (or cytochrome c), TNBS limits the reaction and accepts a total of four electrons. The TNBS-dependent NADPH (or NADH) oxidation is markedly stimulated by NADP+, and to a smaller extent also by NAD+. The TNBS-dependent reactions are inhibited by excess of NADPH but not by NADH. The kinetics of these reactions are consistent with a branching reaction mechanism in which a pathway including a ternary complex between the two-electron reduced enzyme and NADP+ has the highest turnover. NADPH-dependent reductions of ferricyanide or 2,6-dichloroindophenol catalyzed by glutathione reductase are also markedly influenced by NADP+. Evidently NADP+ facilitates a shift of the catalyzed reaction from the normal two-electron reduction of glutathione disulfide to a more unspecific one-electron reduction of other acceptors. Spectral as well as kinetic data suggest that the rate of radical formation limits the reactions with the artificial electron acceptors and that NADP+ promotes this rate-limiting step.     

The carbonate radical is a site-selective oxidizing agent of guanine in double-stranded oligonucleotides

The carbonate radical anion (CO(3)) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO(2) with ONOO(-)) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO(3) anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.     

Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2

The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 degree C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1 = 1.4 x 10(7) M-1 s-1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+.FeIVO]. Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2 = 65 s-1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome-c peroxidase, was assigned to a two-electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+. which was formed by an intramolecular electron transfer from tyrosine to the porphyrin-pi-cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.     

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).     

Horseradish peroxidase-mediated aerobic and anaerobic oxidations of 3-alkylindoles

Little is known about the HRP-mediated oxidations of 3-alkylindoles. Whereas 3-methylindole and 3-ethylindole were found to be turned over smoothly by HRP, reactions of tryptophol and N-acetyltryptamine were inefficient. Oxidations of the former two indoles by HRP under aerobic conditions led to the corresponding ring-opened o-acylformanilides and oxindoles, whereas anaerobic oxidations resulted in oxidative dimerization. The major products of anaerobic oxidation of 3-methylindole were shown to be two hydrated dimers, while anhydrodimers were obtained in the 3-ethyl case. The proposed mechanism involves HRP-mediated one-electron oxidation to give an indole radical that either dimerizes (anaerobic conditions) or reacts with O2 (aerobic conditions). Measured kinetics of indole oxidation by HRP compounds I and II mirrored their relative reactivities under turnover conditions. The observed comparable binding affinities for all four indole substrates investigated suggest that the low reactivity of tryptophol and N-acetyltryptamine reflect binding to HRP in an orientation that is disadvantageous to electron transfer oxidation of the indole ring.     

Stopped-flow investigation of the reaction of vitamin C with tocopheroxyl radical in aqueous triton X-100 micellar solutions. The structure-activity relationship of the regeneration reaction of tocopherol by vitamin C

A kinetic study of the reaction between vitamin C (L-ascorbic acid, AsH2) and a tocopheroxyl radical (7-tert-butyl-5-isopropyltocopheroxyl) in Triton X-100 micellar solution has been performed using stopped-flow spectrophotometry. The second-order rate constants (k2) obtained showed notable pH dependence with a broad maximum around pH 8. For instance, the k2 values obtained were 26 M-1 S-1 at pH 3, 322 M-1 S-1 at pH 7, and 273 M-1 S-1 at pH 10. A good correlation between the rate constants and the mole fraction of ascorbate monoanion (AsH-) was observed, showing that ascorbate (AsH-) can regenerate the tocopherol from tocopheroxyl in biological systems. Furthermore, the results indicate that reduced ascorbic acid (AsH2) does not have the ability to regenerate the tocopherol in aqueous solution. On the other hand, it was found that AsH2 can reduce the tocopheroxyl to tocopherol in benzene/ethanol (2:1) mixtures, although the rate of reaction is only approximately 15% of that observed in micellar solution at pH 7.     

Substituted p-hydroquinones as inhibitors of lipid peroxidation

The technique based on monitoring oxygen consumption was applied to study 12 alkyl- and methoxy-substituted p-hydroquinones (QH(2)) as a chain-breaking antioxidant during the oxidation of styrene and methyl linoleate (ML) in bulk as well as ML oxidation in micellar solution of sodium dodecyl sulfate (SDS) at 37 degrees C. The antioxidant activities of QH(2) were characterized by two parameters: the rate constant k(1) for reaction of QH(2) with the peroxy radical LO(2)*: QH(2)+LO(2)*-->QH*+LOOH and the stoichiometric factor of inhibition, f, which shows how many kinetic chains may be terminated by one molecule of QH(2). In the case of styrene and ML oxidation in bulk, f values never exceed two; for the majority of QH(2), f was found to be significantly less than two due to the interaction of QH* with molecular oxygen. In the absence of superoxide dismutase (SOD), all the studied QH(2) displayed a very moderate if any antioxidant capability during ML oxidation in SDS micelles. When 20U/ml SOD was added, the majority of QH(2) showed a pronounced ability to inhibit ML oxidation, f parameter being ca. one. The features of QH(2) as an antioxidant in aqueous environment are suggested to associate with the reactivity of semiquinone (Q*(-)). Q*(-) reacts readily with molecular oxygen with formation of superoxide (O(2)*(-)); further reactions of O(2)*(-) result in fast depleting QH(2) and chain propagation. The addition of SOD results in purging a reaction mixture from O(2)*(-) and, as a corollary, in depressing undesirable reactions with the participation of O(2)*(-). With all the oxidation models, QH(2) were found to be very reactive to LO(2)*. The rate constants k(1) decreased progressively when going from the oxidation of styrene to ML oxidation in bulk and further to ML oxidation in SDS micelles.