Interpretation of the reactivity of peroxidase compounds I and II with phenols by the Marcus equation.

The catalytic cycle of heme peroxidases involves two reactive states, compound I and compound II. Although their reduction potentials at pH 7 are similar, compound I is in general more reactive towards organic substrates than compound II. The different reactivities have until now remained unexplained. In this study, the reactions of compounds I and II of peroxidase from horseradish with phenols were analyzed using the Marcus equation of electron-transfer. Both reactions exhibit similar reorganization energies, and the different reactivities of the two enzyme states can be ascribed to a higher apparent rate of activationless electron-transfer in the compound I reactions. This can be attributed to the shorter electron-tunneling distance on electron-transfer to the porphyrin radical cation in compound I, compared to electron-transfer to the iron ion in compound II.

Free radical intermediates in the oxidation of flavone-8-acetic acid: possible involvement in its antitumour activity.

The sulphate radical (SO4.-), a model one-electron oxidant, reacts with the antitumour drug flavone-8-acetic acid (FFA) with the rate constant 9.1 x 10(8) dm3mol-1s-1 to yield an uncharged radical that reacts with oxygen (k approximately 1 x 10(9) dm3mol-1s-1). The oxidation of FAA by SO4.- in a steady-state system was found to release carbon dioxide with a yield of 96% relative to that of the SO.4-. The results are interpreted by fast (t1/2 < or = 1 microsecond) and efficient decarboxylation of the FAA radical cation, resulting in a carbon-centred radical. The reaction of the latter with oxygen is a possible source of radical-driven cytotoxic pathways, such as singlet oxygen formation via the Russell mechanism or H-abstraction from lipids. On the basis of the observations in the model system, a possible free radical mechanism for the antitumour action of the drug is suggested.

Free hydroxyl radicals are formed on reaction between the neutrophil-derived species superoxide anion and hypochlorous acid.

Superoxide anion reacts with hypochlorous acid to yield free hydroxyl radicals, as shown by the hydroxylation of benzoate. This reaction is analogous to the Haber-Weiss reaction but in the absence of metal ions is at least six orders of magnitude faster.

Antioxidant activities of carotenes and xanthophylls.

The purpose of this study was to assess the relative antioxidant activities of a range of carotenes and xanthophylls through the extent of their abilities to scavenge the ABTS(.+) radical cation. The results show that the relative abilities of the carotenoids to scavenge the ABTS(.+) radical cation are influenced by the presence of functional groups with increasing polarities, such as carbonyl and hydroxyl groups, in the terminal rings, as well as by the number of conjugated double bonds.

H2O2-driven reduction of the Fe3+-quin2 chelate and the subsequent formation of oxidizing species.

The objective of this study was to compare effects of quin2 and EDTA in iron-driven Fenton-type reactions. Seven different assays for detection of strong oxidants were used: the DMSO, deoxyribose, benzoate hydroxylation, and plasmid DNA strand breakage assays, detection of 8-oxo-deoxyguanosine in deoxyguanosine mononucleosides and calf thymus DNA, and electron spin resonance with the spin-trap (4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in the presence of ethanol or DMSO. With H2O2 and Fe3+, quin2 generally strongly increased the formation of reactive species in all assays, whereas with EDTA the results varied between the assays from barely detectable to highly significant increases compared to H2O2 and unchelated Fe3+. We found that the species produced in the reaction between Fe3+-quin2 and H2O2 behaved like the hydroxyl radical in all assays, whereas with Fe3+-EDTA no clear conclusion could be drawn about the nature of the oxidant. The effect of quin2 on the formation of oxidants on Fe2+ autoxidation, varied from generally inhibiting to slightly promoting, depending on the assay used. EDTA had a promoting effect on the amount of oxidant detected by all but one assay. None of the autoxidation systems produced DMSO or ethanol radical adducts with 4-POBN. In the presence of either chelator, H2O2, and Fe2+ DMSO and ethanol radical adducts of 4-POBN were produced. Using the Fe2+ indicator ferrozine, evidence for direct reduction of Fe3+-quin2 by H2O2 was found. Superoxide anion radical appeared to be less efficient than H2O2 as reductant of Fe3+-quin2 as addition of superoxide dismutase in the ferrozine experiments only decreased the amount of Fe2+ available for Fenton reaction by 10-20%. The main conclusions from our study are that the reduction of Fe3+-quin2 can be driven by H2O2 and that Fe2+ in the following oxidation step produces a species indistinguishable from free hydroxyl radical.

Toward targeted "oxidation therapy" of cancer: peroxidase-catalysed cytotoxicity of indole-3-acetic acids.

PURPOSE: The study aimed to identify suitable prodrugs that could be used to test the hypothesis that peroxidase activity in cells, either endogenous or enhanced by immunological targeting, can activate prodrugs to cytotoxins. We hypothesized that prototype prodrugs based on derivatives of indole-3-acetic acid (IAA), when activated by peroxidase enzymes (e.g., from horseradish, HRP) should produce peroxyl radicals, with deleterious biological consequences. METHODS AND MATERIALS: V79 hamster cells were incubated with IAA or derivatives +/- HRP and cytotoxicity assessed by a clonogenic assay. To assess the toxicity of stable oxidation products, prodrugs were also oxidized by HRP without cells, and the products then added to cells. RESULTS: The combination of prodrug and enzyme resulted in cytotoxicity, but neither indole nor enzyme in isolation was toxic under the conditions used. Although lipid peroxidation was stimulated in liposomes by the prodrug/enzyme treatment, it could not be measured in mammalian cells. Adding oxidized prodrugs to cells resulted in cytotoxicity. CONCLUSIONS: Although the hypothesis that prodrugs of this type could enhance oxidative stress via lipid peroxidation was not established, the results nonetheless demonstrated oxidatively-activated cytotoxicity via indole acetic acid prodrugs, and suggested these as a new type of substrate for antibody-directed enzyme-prodrug therapy (ADEPT). The hypothesized free-radical fragmentation intermediates were demonstrated, but lipid peroxidation associated with peroxyl radical formation was unlikely to be the major route to cytotoxicity.

Peroxidase-catalyzed effects of indole-3-acetic acid and analogues on lipid membranes, DNA, and mammalian cells in vitro.

This study aimed to explore the mechanisms and molecular parameters which control the cytotoxicity of derivatives of indole-3-acetic acid (IAA) when oxidatively activated by horseradish peroxidase (HRP). Lipid peroxidation was measured in liposomes, damage to supercoiled plasmid DNA assessed by gel electrophoresis, free radical intermediates detected by EPR following spin trapping, binding of IAA-derived products demonstrated by 3H labelling, stable products measured by HPLC, and cytotoxicity in hamster fibroblasts measured by clonogenic survival. IAA, and nine analogues more easily oxidized by HRP, caused lipid peroxidation in liposomes, but not detectably in membranes of hamster fibroblasts, and were cytotoxic after HRP activation to varying degrees. Cytotoxicity was not correlated with activation rate. The hydrophilic vitamin E analogue, Trolox, inhibited cytotoxicity, whereas loading fibroblasts with vitamin E was ineffective, consistent with an oxidative mechanism in which radical precursors to damage are intercepted by Trolox in the aqueous phase. However, two known oxidation products were nontoxic (the 3-carbinol and 3-aldehyde, both probably produced from 3-CH2OO* peroxyl radicals via the 3-CH*2 [skatolyl] radical following decarboxylation of the radical cation). The skatolyl radical from IAA was shown by EPR with spin trapping to react with DNA; electrophoresis showed binding to occur. Treatment of hamster fibroblasts with 5-3H-IAA/HRP resulted in intracellular bound 3H. Together with earlier results, the new data point to unknown electrophilic oxidation products, reactive towards intracellular targets, being involved in cytotoxicity of the IAA/HRP combination, rather than direct attack of free radicals, excited states, or membrane lipid peroxidation.

Fenton chemistry: an introduction.

In 1876, Fenton described a colored product obtained on mixing tartaric acid with hydrogen peroxide and a low concentration of a ferrous salt. Full papers in 1894 and 1896 showed the product was dihydroxymaleic acid. Haber, Weiss and Willstätter proposed in 1932-1934 the involvement of free hydroxyl radicals in the iron(II)/hydrogen peroxide system, and Baxendale and colleagues around 1950 suggested that superoxide reduces the iron(III) formed on reaction, explaining the catalytic nature of the metal. Since Fridovich and colleagues discovered the importance of superoxide dismutase in 1968, numerous studies have sought to explain the deleterious effects of cellular oxidative stress in terms of superoxide-driven Fenton chemistry. There remain questions concerning the involvement of free hydroxyl radicals or reactions of metal/oxo intermediates. However, these outstanding questions may obscure a wider appreciation of the importance of Fenton chemistry involving hypohalous acids rather than hydrogen peroxide as the oxidant.

Formation of hydroxyl radicals on reaction of hypochlorous acid with ferrocyanide, a model iron(II) complex.

Hypochlorous acid reacts with the model iron(II) complex, ferrocyanide (Fe(CN)6(4-)) in aqueous solution with the rate constant 220 +/- 15 dm3 mol-1 s-1. Free hydroxyl radicals are formed in this reaction in 27% yield as shown by the hydroxylation of benzoate to give a product distribution identical to that of free (radiolytically generated) hydroxyl radicals. This reaction is three orders of magnitude faster than the analogous reaction involving hydrogen peroxide (the Fenton reaction), suggesting that the hypochlorous acid generated by activated neutrophils may be a source of hydroxyl radicals.

Enhancement of lipid peroxidation by indole-3-acetic acid and derivatives: substituent effects.

The peroxidation of liposomes by a haem peroxidase and hydrogen peroxide in the presence of indole-3-acetic acid and derivatives was investigated. It was found that these compounds can accelerate the lipid peroxidation up to 65 fold and this is attributed to the formation of peroxyl radicals that may react with the lipids, possibly by hydrogen abstraction. The peroxyl radicals are formed by peroxidase-catalyzed oxidation of the enhancers to radical cations which undergo cleavage of the carbon-carbon bond on the side-chain to yield CO2 and carbon-centred radicals that rapidly add oxygen. In competition with decarboxylation, the radical cations deprotonate reversibly from the N1 position. Rates of decarboxylation, pka values and rate of reaction with the peroxidase compound I indicate consistent substituent effects which, however, can not be quantitatively related to the usual Hammett or Brown parameters. Assuming that the rate of decarboxylation of the radical cations taken is a measure of the electron density of the molecule (or radical), it is found that the efficiency of these compounds as enhancers of lipid peroxidation increases with increasing electron density, suggesting that, at least in the model system, the oxidation of the substrates is the limiting step in causing lipid peroxidation.

The reaction of oxygen with radicals from oxidation of tryptophan and indole-3-acetic acid.

The oxidation of tryptophan and indole-3-acetic acid (IAA) by the dibromine radical anion or peroxidase from horseradish in aqueous solution was investigated and compared, especially with respect to the involvement of oxygen and superoxide. Using EPR with spin-trapping, the tryptophanyl radical, generated by either method was found to react with oxygen, although this reaction is too slow to be observed by pulse radiolysis (k < 5 x 10(6) dm3 mol-1 s-1). No superoxide results from this reaction, thus excluding an electron-transfer mechanism and suggesting the formation of a tryptophan peroxyl radical, possibly in a reversible process. These observations imply that in proteins where the tryptophanyl radical exists as a stable species it must either have its reactivity modified by the protein environment or be inaccessible to oxygen. The related molecule LAA is oxidized by either peroxidase or Br2.- to a radical cation that decarboxylates to yield a skatolyl radical. The latter reacts with oxygen to give a peroxyl radical that does not release superoxide. However, O2.- is formed during the peroxidase-catalyzed oxidation of indoleacetic acid. This supports the hypothesis that the peroxidase can act in an oxidase cycle involving ferrous enzyme and compound III, with superoxide as a product.

Photoionization of ferrocytochrome c by 248 nm laser light and the observation of the early stages of ferricytochrome c unfolding in the nanosecond-to-millisecond timescale.

Photolysis of ferrocytochrome c by 248 nm laser light in aqueous solution at pH 7 generates hydrated electrons (eaq-) by a monophotonic process with quantum yield phi = 0.034. Approximately three-quarters of the eaq- originate from the heme, which is converted from the ferrous to the ferric state in < 100 ns. The conformational changes associated with the change in the redox state of cytochrome c are either not detectable spectrophotometrically or complete in < 100 ns. Also, under conditions where ferrocytochrome c is stable but ferricytochrome c is unfolded (3 M guanidine, pH 7, 40 degrees C), photoionization of ferrocytochrome c generated ferricytochrome c with similar quantum yield. Under these conditions, the lifetime of native ferricytochrome c is 67 microseconds; it decays via two intermediates with lambda max > 410 nm, neither of which is the thermodynamically favored, unfolded form. These species are putatively identified as unfolding intermediates with nonnative iron ligands, similar to those found during folding of ferrocytochrome c. The results suggest that unfolding, like folding, proceeds by intrachain diffusion and ligand exchange.

Generation of high-oxidation states of myoglobin in the nanosecond time-scale by laser photoionization.

The 248 nm laser flash photolysis of myoglobin in various redox states (oxy, met and ferryl) in neutral aqueous solution yielded hydrated electrons with concurrent changes in the visible absorption spectrum of the heme. The results could be ascribed to the photoionization of both the peptide and the heme group, in approximately equal yields. The ionization of met- and ferrylmyoglobin was biphotonic, but that of oxymyoglobin was a mixture of mono- and biphotonic processes. Using appropriate electron and radical scavengers, the changes in the heme absorption could be investigated at times > or = 100 ns and were shown to be associated with a +1 increase of the formal oxidation state of the heme. Using this method, the formal iron (V) state of native myoglobin could be spectroscopically characterized for the first time. Its absorption, blue-shifted and less intense relative to the ferryl state, is reminiscent of that of the compound I of peroxidases, which contains a ferryl-oxo (iron[IV]) group and a porphyrin radical cation. On this basis, the same structure is proposed for the formal iron(V) state of native myoglobin. The transition from oxy- to metmyoglobin took approximately 5 microsecond, which may reflect the kinetics of exchange of oxygen with water as ligand. The transitions from the met to the ferryl state, and from ferryl to iron(V) states were faster (approximately 250 ns), consistent with processes that involve proton or electron movements but no changes in the iron coordination state.

Enhancement of peroxidase-induced lipid peroxidation by indole-3-acetic acid: effect of antioxidants.

Indole-3-acetic acid (IAA) enhanced the peroxidase-induced lipid peroxidation in phosphatidylcholine liposomes, as measured by loss of fluorescence of cis-parinaric acid. α-Tocopherol or ß-carotene in the lipid phase or ascorbate or Trolox in the aqueous phase inhibited the loss of fluorescence induced by the peroxidase + IAA system, but glutathione had only a small inhibitory effect. The peroxyl radical formed by one-electron oxidation of IAA, followed by decarboxylation and reaction with oxygen, is suggested to act as the initiator of lipid peroxidation. The protection by ascorbate or Trolox is explained by the reactivity of these compounds with the IAA indolyl radical, as shown by pulse radiolysis experiments, whereas the weak effect of glutathione agrees with its low reactivity towards the IAA-derived peroxyl radical and its precursors.

Induction of strand breaks in polyribonucleotides and DNA by the sulphate radical anion: role of electron loss centres as precursors of strand breakage.

The interaction of the sulphate radical anion, SO4.-, with the polyribonucleotides, poly U and poly C, in deaerated, aqueous solutions at pH 7.5 results in strand breakage (sb) with efficiencies of 57 and 23%, respectively, determined by time resolved laser light scattering (TRLS). Most sb are produced within 70 microseconds, the risetime of the detection system. Oxygen inhibits the induction of sb in poly U and poly C by SO4.- through its interaction with a radical precursor to sb. In contrast, the interaction of SO4.- with poly A and single stranded DNA does not lead to significant strand breakage (< or = 5% efficiency). From optical studies, the interaction of poly A and poly G with SO4.- radicals yields predominantly the corresponding one electron oxidized base radicals. With poly C and poly U, it is proposed that the SO4.- radical interacts predominantly by addition to the base moiety to produce the C(5)-yl and C(6)-yl sulphate radical adducts which react with oxygen. These base adducts subsequently interact with the sugar-phosphate moiety by H-atom abstraction to yield C(2)' sugar radicals with rate constants in the range 1.3-1.7 x 10(5) s-1. It is proposed that the C(2)' sugar radical leads to strand breakage within 70 microseconds, in competition with its transformation into the C(1)'-sugar radical involving base release. From optical studies on the interaction of SO4.- with double stranded DNA, it is suggested that the predominant radical species produced in DNA is the one-electron oxidized radical of guanine, consistent with positive charge migration in DNA. Since the efficiency of SO4.- to induce sb in single stranded DNA is low, it is concluded that the one-electron oxidized guanine radical does not effectively induce strand breakage in DNA.

Radiation chemistry applied to drug design.

Radiation chemistry can contribute to drug design by quantifying redox properties of drugs (useful parameters in quantitative structure-activity relationships), and where free radicals are suspected intermediates in drug action, radiation can be used to generate these putative species and help characterize relevant reactions. Steady radiolysis produces radicals at a readily-varied but quantified rate; pulse radiolysis with fast spectrophotometric and/or conductimetric detection enables the kinetic properties of radicals to be monitored directly. Using these methods, radical intermediates from drugs with specific cytotoxicity towards hypoxic cells have been shown to react rapidly with oxygen, a reaction probably responsible for the therapeutic differential. Radical oxidants from activated neutrophils include superoxide and hydroxyl radicals, and radiation-chemical methods have an important role to play in rational drug design to exploit such oxidative chemistry. Antioxidants can also be evaluated quantitatively by radiolysis methods; the conjugation reactions of thiyl radicals with thiolate and oxygen are now recognised to be major contributions of pulse radiolysis to thiol biochemistry.

Lifetime and reactivity of the veratryl alcohol radical cation. Implications for lignin peroxidase catalysis.

The formation and decay of veratryl alcohol radical cation upon oxidation of veratryl alcohol by thallium (II) ions was studied by pulse radiolysis with spectrophotometric and conductometric detection. In aqueous solution at pH 3 the radical cation decays by a first order process, assigned to the deprotonation from the alpha-carbon. On the basis of its lifetime (59 +/- 8 ms) and of its ability to oxidize a polymeric dye (Poly R-478) we estimate that the radical cation can diffuse about 7 microns in an aqueous environment to act as a mediator of oxidations over long distances. However, 4-methoxymandelic acid is not oxidized by the veratryl alcohol radical cation in homogeneous solution, and the comparison with previous studies on lignin peroxidase catalysis suggests a second role for veratryl alcohol radical cation in the enzyme action: it may exist as an enzyme-bound species that has either a longer lifetime or a higher reduction potential than the free radical cation in bulk solution.

Reaction of HO* with guanine derivatives in aqueous solution: formation of two different redox-active OH-adduct radicals and their unimolecular transformation reactions. Properties of G(-H)*.

The reaction of *OH with 2'-deoxyguanosine yields two transient species, both identified as OH adducts (G*-OH), with strongly different reactivity towards O2, or other oxidants, or to reductants. One of these, identified as the OH adduct at the C-8 position (yield 17% relative to *OH), reacts with oxygen with k=4 x 10(9)M(-1)s(-1); in the absence of oxygen it undergoes a rapid ring-opening reaction (k = 2 x 10(5) s(-1) at pH4-9), visible as an increase of absorbance at 300-310 nm. This OH adduct and its ring-opened successor are one-electron reductants towards, for example, methylviologen or [Fe(III)(CN)6]3-. The second adduct, identified as the OH adduct at the 4-position (yield of 60-70% relative to *OH), has oxidizing properties (towards N,N,N',N'-tetra-methyl-p-phenylenediamine, promethazine, or [Fe(II)(CN)6]4-). This OH adduct undergoes a slower transformation reaction (k = 6 x 10(3) s(-1) in neutral, unbuffered solution) to produce the even more strongly oxidizing (deprotonated, depending on pH) 2'-deoxyguanosine radical cation, and it practically does not react with oxygen (k< or = 10(6)M(-1)s(-1)). The (deprotonated) radical cation, in dilute aqueous solution, does not give rise to 8-oxoguanosine as a product. However, it is able to react with ribose with k< or =4 x 10(3)M(-1)S(-1).

Kinetics and mechanisms of hypochlorous acid reactions.

Hypochlorous acid (HOCl) is a strong oxidant formed in neutrophils by the myeloperoxidase-catalyzed oxidation of chloride. Using stopped-flow with spectrophotometric detection, HOCl was found to react very rapidly with glutathione and ascorbate and less rapidly with taurine. No evidence could be found for the formation of reactive free-radical intermediates in these reactions, in support of an electrophilic mechanism. In contrast, the reaction with iron(II) aquo or citrate complexes (k approximately 10(4) dm3 mol-1 s-1 in acidic solution) yielded reactive intermediates distinguishable from hydroxyl radicals. The reaction between HOCl and ferrous ions, which is analogous to but faster than the Fenton reaction, is a potential source of free radicals in activated neutrophils.