Pulse-radiolytic investigations of catechols and catecholamines. II. Reactions of Tiron with oxygen radical species.

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (.OH), superoxide anions (O-2) or a combination of both oxygen radicals. The rate constant with .OH radicals was determined at 1.0.10(9) M-1.s-1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11--16), the rate constant with O-2 of 1.0.10(7) M-1.s-1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with .OH and O-2 of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of .OH radicals, leading to the same products with catechol compounds.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

Antioxidant properties of nitecapone (OR-462).

Nitecapone [3-(3,4-dihydroxy-5-nitrophenyl)methylene-2,4-pentanedione] [OR-462] is a catechol-O-methyltransferase inhibitor with gastroprotective properties. Recently, its antioxidant properties have been discovered: It scavenges peroxyl radicals (ROO.) and thus spares glutathione. Further examination of the properties of nitecapone demonstrated a remarkable ability of this compound to act as an antioxidant: (1) to scavenge ROO. in solution with a stoichiometry factor of 2; (2) to scavenge ROO. in membranes; (3) to inhibit lipid peroxidation; (4) to act as a competitive inhibitor for xanthine oxidase with Ki of 8.8 microM; (5) to scavenge O2- with a second order kinetic rate constant of 1.0 x 10(4) M-1 s-1; and (6) to scavenge HO.. Nitecapone also interacts with oxidation product of ascorbate to participate in recycling of vitamin E. Thus, nitecapone potentially is an effective therapeutic antioxidant, and the use of this compound in a combination with other antioxidants may be beneficial.     

The oxidation of ferrocytochrome c by Br2-, (SCN)2-, N3 and OH radicals studied by pulsed-electron and gamma-ray radiolysis

The reactions of ferrocytochrome c with Br2-, (SCN)2-, N3 and OH radicals were followed by measuring the change in the optical spectra of cytochrome c on gamma-irradiation as well as the rate of change of absorbance upon pulse irradiation. Ferrocytochrome c is oxidized to ferricytochrome c by Br2-, (SCN)2- or N3 radical with an efficiency of about 100% through a second-order process in which no intermediates were observed. The rate constants in neutral solutions at I = 0.073 are 9.7 . 10(8) M-1 . s-1, 7.9 . 10(8) M-1, 1.3 . 10(9) M-1 . s-1 for the oxidation by Br2-, (SCN)2- and N3 radicals, respectively. The rate constants do not vary appreciably in alkaline solutions (pH 8.9). The ionic strength dependence was observed for the rate constants of the oxidation by br2- and (SCN)2-. Those rate constants estimated on the assumption that the radicals react only with the amino acid residues with the characteristic steric correction factors were less than one-tenth of the observed ones. These results suggest that the partially exposed region of the heme is the probable site of electron transfer from ferrocytochrome c to the radical. Hydroxyl radicals also oxidize ferrocytochrome c with a high rate constant (k greater than 1 . 10(10) M-1 . s-1), but with a very small efficiency (5%).     

Superoxide radical production during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium perchlorate.

1-Methyl-4-phenyl-2,3-dihydropyridinium perchlorate (MPDP+), an intermediate in the metabolism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, was found to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected by their ability to reduce ferricytochrome c. Superoxide dismutase inhibited this reduction in a dose-dependent manner. The rate of reduction of ferricytochrome c was dependent not only on the concentration of MPDP+ but also on the pH of the system. Thus, the rate of autoxidation of MPDP+ and the sensitivity of this autoxidation to superoxide dismutase-inhibitable ferricytochrome c reduction were both augmented, as the pH was raised from 7.0 to 10.5. The rate constant (Kc) for the reaction of superoxide radical with ferricytochrome c to form ferricytochrome c was found to be 3.48 x 10(5) M-1 s-1. The rate constant (KMPDP+) for the reaction of MPDP+ with ferricytochrome3+ c was found to be only 4.86 M-1 s-1. These results, in conjunction with complexities in the kinetics, lead to the proposal that autoxidation of MPDP+ proceeds by at least two distinct pathways, one of which involves the production of superoxide radicals and hence is inhibitable by superoxide dismutase. It is possible that the free radicals so generated could induce oxidative injury which may be central to the MPTP/MPDP(+)-induced neuropathy.     

Inhibition of oxidation of methyl linoleate in solution by vitamin E and vitamin C

The oxidation of methyl linoleate in solution initiated with azo compounds has been studied in the absence and presence of vitamin E and vitamin C. Both vitamin E and vitamin C acted as a chain-breaking antioxidant and they suppressed the oxidation and produced an induction period. The inhibition rate constant for the scavenging of peroxy radical was calculated at 37 degrees C as kinh = 5.1 X 10(5) M-1 s-1 and 7.5 X 10(4) M-1 s-1 for vitamin E and vitamin C, respectively. It was suggested that each vitamin E could trap two peroxy radicals, whereas vitamin C could trap only one peroxy radical under the reaction conditions employed in this study. When both vitamin E and vitamin C were present, the oxidation was suppressed quite efficiently and the apparent inhibition rate constant was obtained as kinh = 4.0 X 10(5) M-1 s-1. Furthermore, vitamin E remained almost unchanged and only vitamin C was consumed at the initial stage and vitamin E was consumed after vitamin C was exhausted. It was concluded that vitamin E trapped the peroxy radical and the resulting alpha-chromanoxy radical reacted with vitamin C to regenerate vitamin E.     

Chemically induced Parkinson's disease. II: Intermediates in the oxidation and reduction reactions of the 1-methyl-4-phenyl-2,3-dihydropyridinium ion and its deprotonated form

The one-electron reduction product of 1-methyl-4-phenyl-2,3-dihydropyridinium ion has been generated by pulse radiolysis and its absorption spectrum recorded. This radical was found to decay by second-order kinetics (2k = 9.5 x 10(8) M-1 s-1) to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-2,3-dihydropyridinium ion. Reactions of the above radical species and that formed by one-electron reduction of 1-methyl-4-phenylpyridinium ion, which can also be generated by one-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, with a number of molecules of biochemical interest have been studied. The one-electron reduction product of oxidised nicotinamide adenine dinucleotide efficiently reduced 1-methyl-4-phenyl-2,3-dihydropyridinium ion (k = 2.2 x 10(9) M-1 s-1). The relevance of these results in relation to redox cycling, a possible mechanism for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity, is discussed.     

Evaluation of the ability of the angiotensin-converting enzyme inhibitor captopril to scavenge reactive oxygen species.

Captopril, an inhibitor of angiotensin-converting enzyme, has been suggested to have additional cardioprotective action because of its ability to act as an antioxidant. The rates of reaction of captopril with several biologically-relevant reactive oxygen species were determined. Captopril reacts slowly, if at all, with superoxide (rate constant less than 10(3) M-1 s-1) or hydrogen peroxide (rate constant less than M-1 s-1). It does not inhibit peroxidation of lipids stimulated by iron ions and ascorbate or by the myoglobin/H2O2 system. Indeed, mixtures of ferric ion and captopril can stimulate lipid peroxidation. Captopril reacts rapidly with hydroxyl radical (rate constant greater than 10(9) M-1 s-1) but might be unlikely to compete with most biological molecules for OH because of the low concentration of captopril that can be achieved in vivo during therapeutic use. Captopril did not significantly inhibit iron ion-dependent generation of hydroxyl radicals from hydrogen peroxide. By contrast, captopril is a powerful scavenger of hypochlorous acid: it was able to protect alpha 1-antiproteinase (alpha 1 AP) against inactivation by this species and to prevent formation of chloramines from taurine. We suggest that the antioxidant action of captopril in vivo is likely to be limited, and may be restricted to protection against damage by hypochlorous acid derived from the action of neutrophil myeloperoxidase.     

Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium. Role of veratryl alcohol in polymer oxidation.

The mechanism of lignin peroxidase (LiP) was examined using bovine pancreatic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE analysis demonstrates that an RNase dimer is the major product of the LiP-catalyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP-catalyzed oxidation of Tyr residues to Tyr radicals, followed by intermolecular radical coupling. The LiP-catalyzed polymerization of RNase in strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 microM H2O2, relatively low concentrations of RNase and VA, together but not individually, can protect LiP from H2O2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped-flow and rapid-scan spectroscopy demonstrate that the reduction of LiP compound I (LiPI) to the native enzyme by RNase occurs via two single-electron steps. At pH 3.0, the reduction of LiPI by RNase obeys second-order kinetics with a rate constant of 4.7 x 10(4) M-1.s-1, compared to the second-order VA oxidation rate constant of 3.7 x 10(5) M-1.s-1. The reduction of LiP compound II (LiPII) by RNase also follows second-order kinetics with a rate constant of 1.1 x 10(4) M-1.s-1, compared to the first-order rate constant for LiPII reduction by VA. When the reductions of LiPI and LiPIi are conducted in the presence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.     

Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 degrees C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant k2,app, was found to fall within the range of 5.8 x 10(6) +/- 0.4 x 10(6) M-1.s-1 less than k2,app less than 1.8 x 10(7) +/- 0.1 x 10(7) M-1.s-1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme-substrate complex formation, with kcat = 22 +/- 3 s-1, Km = 67 +/- 10 microM, and the second-order rate constant k3,app = 2.0 x 10(5) +/- 0.2 x 10(5) M-1.s-1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 +/- 0.5 s-1, Km = 49 +/- 7 microM, and k3,app = 1.03 x 10(5) +/- 0.07 x 10(5) M-1.s-1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 +/- 5 and 0.52 +/- 0.05 s-1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 +/- 5 and 0.33 +/- 0.02 s-1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stopped-flow investigation of antioxidant activity of estrogens in solution

A kinetic study of the reaction between estrogens (female hormone) and substituted phenoxyl radical has been performed, as a model for the reactions of estrogens with lipid peroxyl radical in biological systems. The rates of reaction of estrogens (estrone 1, estradiol 2, 2-methoxyestrone 3, 3-methoxyestrone 4, and 2-hydroxyestrone 5) with substituted phenoxyl radical in benzene have been determined spectrophotometrically, using stopped-flow technique. The second-order rate constants, k2, obtained are 84 M-1.s-1 for 1, 138 M-1.s-1 for 2, 520 M-1.s-1 for 3, less than 10(-4) M-1.s-1 for 4, and 2.6 X 10(5) M-1.s-1 for 5 at 25.0 degrees C. 2-Hydroxyestrone 5 was found to be 2.9-times more active than alpha-tocopherol, which has the highest antioxidant activity among natural tocopherols. The order of magnitude of k2 value (1 less than 2 less than 3 less than alpha-Toc less than 5) is in agreement with that of in vitro tests of their antioxidant activities, as measured by the inhibition of lipid peroxidation. Further, similar measurements have been performed for the reaction between the above estrogens 1-5 and tocopheroxyl 6 in benzene solution. It was found that the estrogens having an OH group at the aromatic ring have an ability to regenerate the tocopheroxyl 6 to tocopherol. Especially, the 2-hydroxyestrone 5 showed about three orders of magnitude higher reactivity than ascorbic acid.     

Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.     

Albumin-bound quercetin repairs vitamin E oxidized by apolipoprotein radicals in native HDL3 and LDL

In the minor fraction of HDL3 containing alpha-tocopherol (alphaTocOH), selective one-electron oxidation of Trp and Tyr residues of apolipoproteins A-I and A-II by *Br2- radical-anions produces the corresponding semioxidized species, TyrO* and *Trp. Repair of TyrO* by endogenous alphaTocOH generates the alpha-tocopheroxyl radical (alphaTocO*). Fast spectroscopic studies show that two populations representing 80% of alphaTocO* initially formed are repaired over several seconds with rate constants of 3.0 x 10(6) and 1.5 x 10(5) M-1 s-1 by quercetin bound to human serum albumin (HSA) at physiologically relevant concentration. Formation of HSA-bound quercetin radicals (*Qb) is observed. In the major fraction of HDL3 particles lacking alphaTocOH, TyrO* and *Trp are repaired by free and HSA-bound quercetin. In LDL particles which all contain alphaTocOH, alphaTocO* radicals are formed in the millisecond time scale by repair of TyrO* radicals produced in apolipoprotein B. Then, 75% of initial alphaTocO* are repaired over seconds by HSA-bound quercetin (rate constant: 2.0 x 10(6) M-1 s-1). HSA-bound quercetin can also repair *Trp radicals. In O2-saturated solutions, the fraction of alphaTocO* radicals (more than 50%) not repaired by superoxide radical-anions can be repaired by HSA-bound quercetin with formation of *Qb but to a much lesser extent in LDL than in HDL.     

Characterisation and quantitation of a selenol intermediate in the reaction of ebselen with thiols.

The reaction of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) with thiols was investigated with particular attention to the formation of an ebselen selenol intermediate. The selenol intermediate could be trapped in a mixture of ebselen and thiols with 1-chloro-2,4-dinitrobenzene and the resulting product displayed unique spectral characteristics. The reaction of authentic, synthesised ebselen selenol with 1-chloro-2,4-dinitrobenzene (CDNB) was shown to give rise to the same compound (2,4-dinitrophenyl (N-phenyl-2-carboxamido phenyl) selenide as characterized by light spectroscopy, NMR, IR and elemental analysis. The determination of the absorbtion coefficient at 400 nm (E = 7.5 mM-1 cm-1) and the initial rate constant of the reaction (1.4 +/- 0.3 mM-1 min-1) allows for the convenient quantification of ebselen selenol concentrations by initial rate measurements after addition of CDNB. The choice of 400 nm to monitor the reaction excludes the interference of other intermediates in the reaction of ebselen with thiols as well as the reaction of the thiols with CDNB. When the assay is applied to typical incubation conditions used for investigating the glutathione peroxidase-like activity of ebselen it was shown that as much as 10-20% of ebselen is in the selenol form. If a stronger reductant (dithiothreitol) is used 60% is in the selenol form. These data could also be confirmed by the direct determination of ebselen selenol by UV spectroscopy, due to its peak absorption at 370 nm (E = 2 mM-1 cm-1). In conclusion, this investigation demonstrates, for the first time, the identity and quantity of ebselen selenol in the reaction of ebselen with thiols and also describes a convenient assay for its quantification. These observations allow further possibilities for investigation of the molecular species responsible for the antioxidant and peroxidase activities of ebselen.     

Horseradish peroxidase. XXXIV. Oxidation of compound II to I by periodate and inorganic anion radicals.

The one-electron oxidation of horseradish peroxidase compound II to compound I by sodium periodate was observed. The bimolecular rate constant for the NaIO4--compound II interaction is equal to 9.5 +/- 1 x 10(-3) M-1s-1 at room temperature. Irradiation, using ultraviolet light, of the solution containing compound II and persulfate in the presence of bicarbonate, chloride, or bromide, leads ot the fast accumulation of compound I due to the oxidative action of SO4, CO3, Cl2, and Br2 anion radicals, which are products of the photolysis.     

Action of DCFH and BODIPY as a probe for radical oxidation in hydrophilic and lipophilic domain

Fluorogenic probes such as 2',7'-dichlorofluorescin (DCFH) have been extensively used to detect oxidative events and to measure antioxidant capacity. At the same time, however, the inherent drawbacks of these probes such as non-specificity towards oxidizing species have been pointed out. The present study was carried out to analyze the action and dynamics of 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY) and DCFH as a fluorescent probe in the free radical-mediated lipid peroxidation in homogeneous solution, aqueous suspensions of liposomal membranes and LDL and plasma. The rate constant for the reaction of BODIPY with peroxyl radicals was estimated as 6.0 x 10(3) M(-1) s(-1), which makes BODIPY kinetically an inefficient probe especially in the presence of potent radical-scavenging antioxidants such as tocopherols, but a convenient probe for lipid peroxidation. On the other hand, the reactivity of DCFH toward peroxyl radicals was as high as Trolox, a water-soluble analogue of alpha-tocopherol. Thus, DCFH is kinetically more favored probe than BODIPY and could scavenge the radicals within lipophilic domain as well as in aqueous phase. The partition coefficients for BODIPY and DCFH were obtained as 4.57 and 2.62, respectively. These results suggest that BODIPY may be used as an efficient probe for the free radical-mediated oxidation taking place in the lipophilic domain, especially after depletion of alpha-tocopherol, while it may not be an efficient probe for detection of aqueous radicals.     

Action of DCFH and BODIPY as a Probe for Radical Oxidation in Hydrophilic and Lipophilic Domain

Fluorogenic probes such as 2',7'-dichlorofluorescin (DCFH) have been extensively used to detect oxidative events and to measure antioxidant capacity. At the same time, however, the inherent drawbacks of these probes such as non-specificity towards oxidizing species have been pointed out. The present study was carried out to analyze the action and dynamics of 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY) and DCFH as a fluorescent probe in the free radical-mediated lipid peroxidation in homogeneous solution, aqueous suspensions of liposomal membranes and LDL and plasma. The rate constant for the reaction of BODIPY with peroxyl radicals was estimated as 6.0×10₃ M_-1s_-1, which makes BODIPY kinetically an inefficient probe especially in the presence of potent radical-scavenging antioxidants such as tocopherols, but a convenient probe for lipid peroxidation. On the other hand, the reactivity of DCFH toward peroxyl radicals was as high as Trolox, a water-soluble analogue of α-tocopherol. Thus, DCFH is kinetically more favored probe than BODIPY and could scavenge the radicals within lipophilic domain as well as in aqueous phase. The partition coefficients for BODIPY and DCFH were obtained as 4.57 and 2.62, respectively. These results suggest that BODIPY may be used as an efficient probe for the free radical-mediated oxidation taking place in the lipophilic domain, especially after depletion of α-tocopherol, while it may not be an efficient probe for detection of aqueous radicals.     

Nicotinamide adenine dinucleotide species in the horseradish peroxidase-oxidase oscillator.

NADH chemistry ancillary to the oscillatory peroxidase-oxidase (PO) reaction has been reexamined. Previously, (NAD)2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD)2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD)2 can convert horseradish peroxidase (HRP) compound I (CpI) to compound II (CpII) with apparent rate constant (2.7 +/- 0.2) x 105 M-1.s-1 and CpII to HRP at 1 x 105 M-1.s-1. Moreover, a reduction of HRP compound III (CpIII) to CpI by (NAD)2 occurs with a rate constant faster than 5 x 106 M-1.s-1. The (NAD)2 reduction of CpIII provides an alternative to the reduction by NAD radical suggested by Yokota and Yamazaki. HRP catalyzes oxidation of alpha-NADH, not only the beta anomer as previously assumed. Rate constants of alpha- and beta-NADH reactions with CpI are (7.4 +/- 0.4) x 105 M-1.s-1, and (1.7 +/- 0.2) x 105 M-1.s-1, and with CpII are estimated as 5 x 104 M-1.s-1, and 4 x 104 M-1.s-1. Apparent rate constants of reduction of methylene blue (MB) to leuco-methylene blue (MBH) are 3.8 x 104 M-1.s-1 for NADH and 6.4 x 104 M-1.s-1 for NAD dimer, (NAD)2, while reoxidation of MBH proceeds at (2.1 +/- 0.2) x 103 M-1.s-1 All the rates were measured in 0.1 M acetate buffer, pH 5.1.     

NADH oxidation by quinone electron acceptors

The rate constants of NADH oxidation by quinones are increased with the oxidation potential increase: log kox (M-1 X s-1) = -0.25 + 12.2 E0(7) (V) for o-quinones and log kox (M-1 X s-1) = -3.06 + 13.5 E0(7) (V) for p-quinones (pH 7.0, 25 degrees C). It is assumed that the oxidation proceeds via the hydride-ion transfer. The rate constants of NADH oxidation by single-electron quinone acceptors are also increased with the oxidizer potential increase; log kox (M-1 X s-1) = -0.64 + 9.34 E0(7) (V) and correlate with the constants of NADH oxidation by quinone radicals obtained earlier (Grodkowski, J., Neta, P., Carlson, B.W. and Miller, L. (1983) J. Phys. Chem. 87, 3135-3138). Single-electron transfer is the limiting stage of the process.     

A synthetic cysteine oxidase based on a ferrocene-cyclodextrin conjugate.

We report a novel synthetic cysteine oxidase consisting of a ferrocene-beta-cyclodextrin conjugate in which the ferrocene moiety is bound to the secondary hydroxyl side of the cyclodextrin cavity through an ethylenediamine linker. Cysteine oxidation occurs after the ferrocene group is electrochemically oxidized to the ferricinium form, and this generates a voltammetric electrocatalytic wave, the magnitude of which is related to the rate constant for cysteine oxidation. Comparison of cysteine oxidation rates for the primary and secondary beta-cyclodextrin derivatives (105 and 1470 M-1 s-1, respectively) shows that the secondary derivatives are more effective synthetic enzymes. Substrate selectivity of the secondary derivative is demonstrated by comparison of oxidation rates for cysteine (1470 M-1 s-1) and glutathione (260 M-1 s-1) at pH 7.0. The rate constant for cysteine oxidation was 3-fold higher at pH 8.0. With a constant synthetic enzyme concentration, electrocatalytic limiting currents increased linearly with increasing cysteine concentration to a maximum at 6 mM cysteine; above this concentration, the current decreased significantly. These and other results suggest that product inhibition of the catalytic cycle occurs as a result of cystine binding more strongly to the cyclodextrin than cysteine.