Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g^-1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8-9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a K_m of 6.6 mM for H₂O₂ and V_max of 707 mM H₂O₂ min^-1 g^-1 wet wt. cells, and showed saturation kinetics at 50 mM H₂O₂. The cells were entrapped in calcium alginate and used for H₂O₂ degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.     

A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

1. Single reduced methyl viologen (MV^.+) acts as an electron donor in a number of enzyme systems. The large changes in extinction coefficient upon oxidation (λ_max 600 nm; MV^.+, = 1.3 · 10⁴ M⁻¹ · cm⁻¹; oxidised form of methyl viologen (MV²⁺), = 0.0) make it ideally suited to kinetic studies of electron transfer reactions using stopped-flow and standard spectrophotometric techniques.

2. A convenient electrochemical preparation of large amounts of MV^.+ has been developed.

3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.

4. The reaction of MV^.+ with O₂ produced H₂O₂ (k > 5 · 10⁶ M⁻¹ · s⁻¹, pH 7.5, 25 °C). H₂O₂ subsequently reacted with excess MV^.+ (k = 2.3 · 10³ M⁻¹ · s⁻¹, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.

5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H₂O₂ (or radicals derived from H₂O₂) induced damage of plant cell membrane.     

Studies of the oxovanadium(IV)—oxalate system: syntheses and crystal structures of binuclear (Ph4P)2[(VO)2(H2O)2(C2O4)3]·4H2O and of the one-dimensional chain material, (Ph4P)[VOCl(C2O4)]

The hydrothermal reactions of (Ph₄P)[VO₂Cl₂] and H₂C₂O₄ at 150 and 125°C yield (Ph₄P)₂[V₂O₂(H₂O)₂(C₂O₄)₃]·4H₂O (1) and (Ph₄P)[VOCl(C₂O₄)] (2), respectively. The structure of the molecular anion of 1 consists of a binuclear unit of oxovanadium(IV) octahedra bridged by a bisbidentate oxalate group. The VO₆ coordination geometry at each vanadium site is defined by a terminal oxo group, an aquo ligand, and four oxygen donors — two from the bisbidentate bridging oxalate and two from the terminal bidentate oxalate. The structure of 2 consists of discrete Ph₄P⁺ cations occupying regions between [VOCl(C₂O₄)]⁻_∞ spiral chains. The structure of the one-dimensional anionic chain exhibits V(IV) octahedra bridged by bisbidentate oxalate groups. Crystal data: 1·4H₂O, monoclinic P2₁/n, A = 12.694(3), B = 12.531(3), C = 17.17(3) Å, β = 106.32(2)°, V = 2621.3(13) ų, Z = 2, D_calc = 1.501 g cm⁻³, structure solution and refinement converged at a conventional residual of 0.0518; 2, tetragonal P4₃, A = 12.145(2), C = 15.991(3) Å, V = 2358.7(12) ų, Z = 4, R = 0.0452.     

Ascorbate and H2O2 induced oxidative DNA damage in Jurkat cells

The effect of vitamin C (ascorbate) on oxidative DNA damage was examined by first incubating cells with dehydroascorbate, which boosts the intracellular concentration of ascorbate, and then exposing cells to H₂O₂. Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2′-deoxycytidine (oh⁵dCyd) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo⁸dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H₂O₂ (200 μM), resulted in a remarkable sensitization of oh⁵dCyd from 2.7 ± 0.6 to 40.8 ± 6.1 lesions /10⁶ dCyd (15-fold). In contrast, the level of oxo⁸dGuo increased from 8.4 ± 0.4 to 12.1 ± 0.5 lesions/10⁶ dGuo (50%). The formation of oh⁵dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H₂O₂. Additional studies showed that replacement of H₂O₂ with tert-butyl hydroperoxide completely abolished damage, and that preincubation with iron and desferroxamine increased and decreased this damage, respectively. The latter studies suggest that a Fenton reaction is involved in the mechanism of damage. In conclusion, we report a novel model system in which ascorbate sensitizes H₂O₂-induced oxidative DNA damage in cells, leading to elevated levels of oh⁵dCyd and oxo⁸dGuo, with a strong bias toward the formation of oh⁵dCyd.     

Systematic synthesis, structural characterization, and reactivity studies of vanadium(V)–citrate anions [VO2(C6H6O7)]2, isolated from aqueous solutions in the presence of different cations

The aqueous chemistry of vanadium with physiologically relevant ligands constitutes a subject of burgeoning research, extending from bacterial metalloenzymic functions to human-health physiology. Vanadium, in the form of VCl₃ and V₂O_5, reacted expediently with citric acid, in a 1:2 molar ratio in water at pH4, and, in the presence of various cations, afforded crystalline materials bearing the general formula (Cat)₂[V₂O₄(C₆H₆O₇)₂]·nH₂O (A) (Cat⁺=Na⁺, NH₄ ⁺, n=2; Me₄N⁺, K⁺, n=4). Exploration of the reactivity of A toward H₂O₂ yielded the peroxo-containing complexes (Cat)₂[V₂O₂(O₂)₂(C₆H₆O₇)₂]·2H₂O (B) (Cat⁺=K⁺, NH₄ ⁺). Both classes of compounds were characterized analytically and spectroscopically. The X-ray structures of complexes A and B emphasize the exceptional stability of the dimeric rhombic unit V^V ₂O₂, which is retained upon H₂O₂ reaction, and the preserved mode of coordination of the citrate ligand as a doubly deprotonated moiety. In these complexes, typical six and eight coordination numbers were observed for the Na⁺ and K⁺ counter-ions, respectively. The variety of synthetic approaches leading to A, along with the stepwise and direct assembly and isolation of peroxo-compounds (B), denotes the significance of reaction pathways and intermediates in vanadium(III–V)–citrate synthetic chemistry. Hence, a systematic investigation of reactivity modes in aqueous vanadium–citrate systems emerges as a crucial tool for the establishment of chemical interconnectivity among low MW complex species, potentially participating in the intricate biodistribution of that metal ion in biological fluids.     

Two new multi-cobalt-containing heteropolyoxotungstates

Two new multi-cobalt-containing polyoxotungstates K₄Na₆Co₂(H₂O)₁₂{Co(H₂O)₄[Co₂(H₂O)₁₀Co₄(H₂O)₂(B--SiW₉O₃₄)₂]₂} · 40H₂O (1) and K₁₀Na₂[Co₄(H₂O)₂(GeW₉O₃₄)₂] · 20H₂O (2) have been obtained by the routine synthetic reactions in aqueous solution. The polyoxoanion framework of 1 consists of two sandwich-type polyoxoanions [Co₄(H₂O)₂(B--SiW₉O₃₄)₂]¹²⁻ connected together by a [CoO₂(H₂O)₄] cluster to constitute the sandwich dimer, and then, four isolated Co(H₂O)₅ cations coordinate to the dimer through four μ₂-O atoms. The polyoxoanion 2 is isomorphic to the sandwich-type polyoxoanion [Co₄(H₂O)₂(B--SiW₉O₃₄)₂]¹²⁻ in 1. The magnetic property of compound 1 has been studied by measuring its magnetic susceptibility in the temperature range 2.0–300.0 K, indicating the existence of intramolecular ferromagnetic Co–Co interactions, and, the electrochemical properties of 1 and 2 are detected in the pH 4 buffer solution.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

Influence of different site symmetries of Eu centers on the luminescence properties of Anderson-based compounds

Two compounds, [Eu(H₂O)₇][Al(OH)₆Mo₆O₁₈] · 4H₂O (1) and {(C₂H₅NO₂)₂[Eu(H₂O)₅]}[Al(OH)₆Mo₆O₁₈] · 10H₂O (2), have been synthesized by conventional solution method and determined by single-crystal X-ray diffraction. Compound 1 shows a 1D chain structure built up of alternating Anderson-type polyanions [Al(OH)₆Mo₆O₁₈]³⁻ and hydrated rare-earth ions Eu³⁺. Compound 2 displays a 3D supramolecular network structure containing 1D sandglass-like channels along c axis, which were occupied by repetitive array of (H₂O)₈ clusters. Extensive hydrogen bonds play an important role in the formation of the 3D structures of 1 and 2. Luminescence measurements reveal that 1 and 2 exhibit intense red and orange fluorescent emission at room temperature, respectively. Origin of the distinct emission can be assigned to the different site symmetries of Eu³⁺ centers in the two compounds. These results are consistent with the crystal structures of the two compounds.     

Cu2+-catalyzed oxidative degradation of thyroglobulin

Thyroglobulin (Tg) was subjected to metal-catalyzed oxidation, and the oxidative degradation was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. In contrast to no effect of hydrogen peroxide (H₂O₂) alone on the Tg degradation, the inclusion of Cu²⁺ (30 μM), in combination with 2 mM H₂O₂, caused a remarkable degradation of Tg, time- and concentration-dependent. The action of Cu²⁺ was not mimicked by Fe²⁺, suggesting that Tg may interact selectively with Cu²⁺. A similar degradation of Tg was also observed with Cu²⁺corbate system, and the concentration of Cu²⁺ (5-10 μM), in combination with ascorbate, required for the effective degradation was smaller than that of Cu²⁺ (10-30 μM) in combination with H₂O₂. In support of involvement of H₂O₂ in the Cu²⁺ corbate action, catalase expressed a complete protection. However, hydroxyl radical scavengers such as dimethylsulfoxide or mannitol failed to prevent the oxidation of Tg whereas phenolic compounds, which can interact with Cu²⁺, diminished the oxidative degradation, presumably consistent with the mechanism for Cu²⁺-catalyzed oxidation of protein. Moreover, the amount of carbonyl groups in Tg was increased as the concentration (3-100 μM) of Cu²⁺ was enhanced, while the formation of acid-soluble peptides was not remarkable in the presence of Cu²⁺ up to 200 μM. In further studies, Tg pretreated with heat or trichloroacetic acid seemed to be somewhat resistant to Cu²⁺-catalyzed oxidation, implying a possible involvement of protein conformation in the susceptibility to the oxidation. Based on these observations, it is proposed that Tg could be degraded non-enzymatically by Cu²⁺-catalyzed oxidation.     

Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity

The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe²⁺-H₂O₂ or Fe³⁺-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)⁺, only Fe²⁺-H₂O₂ also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe²⁺ iron (concentration range 3-180 μM) or H₂O₂ (concentration range 50-1000 μM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)⁺, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)⁺/ADP-ribose(P) ratio was about 4 at any Fe²⁺ or H₂O₂ concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)⁺/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)⁺ production of Fe²⁺-H₂O₂-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where “conventional” oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.     

Kinetics and mechanism of iron exchange in hydroxamate siderophores: Catalysis of the iron(III) transfer from ferrioxamine B to ethylenediaminetetraacetic acid

The oxalate catalyzed iron(III) transfer from a trihydroxamate siderophore ferrioxamine B, [Fe(Hdfb)⁺], to ethylenediaminetetraacetic acid (H₄edta) has been studied spectro-photometrically in weakly acidic aqueous solutions at 298 K and a constant 2.0 M ionic strength maintained by NaClO₄. The results reveal that oxalate is a more efficient catalyst than the so far studied synthetic monohydroxamic acids. Any role of reduction of Fe(Hdfb)⁺ by oxalate in the catalysis has been rejected by the experimentally observed preservation of the oxalate concentration during the reaction time. Therefore, catalysis has been proposed to be a substitution based process. Under our experimental conditions Fe(Hdfb)⁺ is hexacoordinated and addition of oxalate results in the formation of Fe(H₂dfb)(C₂O₄), Fe(H₃dfb)(C₂O₄)⁻₂ and Fe(C₂O₄)³⁻₃. Therefore, catalysis was proposed to be accomplished by the intermediate formation of the ternary and tris(oxalato) complexes. All three complexes react with H₂edta²⁻ to form thermodynamically stable Fe(edta)⁻ as a final reaction product. Whereas the formation of the ternary complexes is fast enough to feature a pre-equilibrium process to the iron exchange reaction, the formation of Fe(C₂O₄)³⁻₃ is slow and is directly involved in the rate determining step of the Fe(edta)⁻ formation. Nonlinear dependencies of the rate constant on the oxalate and the proton concentrations have been observed and a four parallel path mechanism is proposed for the exchange reaction. The rate and equilibrium constants for the various reaction paths were determined from the kinetic and equilibrium study involving the desferrioxamine B- (H₄dfb⁺), oxalate- and proton-concentration variations. The observed proton catalysis was attributed to the fast monoprotonation of ferrioxamine B as well as of the oxalate ligand. The observed catalysis of iron dissociation from the siderophore has been discussed in view of its significance with respect to in vivo microbial iron transport.     

Enhancing effect of melatonin on chemiluminescence accompanying decomposition of hydrogen peroxide in the presence of copper

The oxidation of melatonin (MEL) using the Cu(II) + H₂O₂ + HO⁻ (the Fenton-like reaction) system was investigated by chemiluminescence (CL), fluorescence, spectrophotometric, and EPR spin trapping techniques. The reaction exhibits CL in the 400–730 nm region. The light emission from the Fenton-like reaction was greatly enhanced in the presence of MEL and was strongly dependent on its concentration. The spectrum measured with cut-off filters revealed maxima at around 460, 500, 580–590, 640–650, and 690–700 nm. The band at 460 nm may be due to the excited cleavage product, N¹-acetyl-N²-formyl-5-methoxykynuramine, whereas the bands at 500, 580–590, 640–650, and 700 nm were similar to those observed for singlet molecular oxygen (¹O₂). The effect of reactive oxygen species (ROS) scavengers on the light emission was studied. The CL was strongly inhibited by the ¹O₂ scavengers in a dose-dependent manner; at concentration 1 mM the potency of ¹O₂ scavenging was 5,5-dimethylcyclohexandione-1,3 > methionine > histidine > hydroquinone. The potency of HO^• scavenging by thiourea, tryptophan, cysteine at concentration 5 mM was 79–94%, by 1 mM glutathione and trolox 75 and 94%, respectively, and by 10 mM cimetidine 18%. Specific acceptors of O₂^•− such as p-nitroblue tetrazolium chloride and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) at concentration 5 mM decreased the CL by 51 and 95%, respectively, whereas superoxide dismutase (SOD) does not reduce the emission at concentration 2.8 U/ml. At higher concentration SOD substantially enhanced the light emission. Addition of 1360 U/ml catalase and 100 μM desferrioxamine strongly inhibited CL (96 and 90%, respectively). The increased generation of ¹O₂ from the Cu/H₂O₂ system in the presence of MEL was confirmed using the spectrophotometric method based on the bleaching of p-nitrosodimethylaniline and by trapping experiments with 2,2,6,6-tetramethylpiperidine (TEMP) and subsequent electron paramagnetic (EPR) spectroscopy. These findings suggest the increased production of reactive oxygen species (O₂^•−, HO^•, ¹O₂) from the Fenton-like reaction in the presence of MEL. This means that the hormone is not able to act as classical chain-breaking antioxidant even at low concentration, and may show clear prooxidant activity at higher concentrations. In addition, long-lived carbonyl product of the MEL transformation in the triplet state can also be toxic by transferring its energy to organelles and causing a photochemical process.     

Kinetics of Reduction of Hypervalent Iron in Myoglobin by Crocin in Aqueous Solution

Crocin in aqueous solution is oxidized by ferrylmyoglobin, MbFe(IV)=O, in a second order reaction with k = 183 1 · mol^-1 · s^-1, AH^‡₂₉₈ = 55.0 kJ · mol^-1, and ΔLS^‡₂₉₈ = -17 J · mol^-1 K^-1 (pH = 6.8, ionic strength 0.16 (NaCl), 25°C), as studied by stopped-flow spectroscopy. The reaction has 1:1 stoichiometry to yield metmyoglobin, MbFe(III), and has AG^o = -11 kJ · mol^-1, as calculated from the literature value E⁰ = +0.85 V (pH = 7.4) vs. NHE for MbFe(IV)=O/MbFe(III) and from the half-peak potential +0.74 V (vs. NHE in aqueous 0.16 NaCl, pH = 7.4) determined by cyclic voltammetry for the one-electron oxidation product of crocin, for which a cation radical structure is proposed and which has a half-peak potential of +0.89 V for its formation from the two-electron oxidation product of crocin. The fer-rylmyoglobin protein-radical, MbFe(IV)=O, reacts with crocin with 2:l stoichiometq to yield MbFe(IV)= 0, as determined by ESR spectroscopy, in a reaction faster than the second order protein-radical generating reaction between H₂O₂ and MbFe(III), for which latter reaction k = 137 L · mol^-1 · s^-1, ΔH^‡₂₉₈ = 51.5 kJ · mol^-1, and ΔH^‡₂₉₈ = -31 J · mol^-1 · K^-1 (pH = 6.8, ionic strength = 0.16 (NaCI), 25°C) was determined. Based on the difference between the stoichiometry for the reaction between crocin and each of the two hypervalent forms of myoglobin, it is concluded in agreement with the determined half peak reduction potentials, that the crocin cation radical is less reducing compared to crocin, as the cation radical can reduce the protein radical but not the iron(IV) centre in hypervalent myoglobin.     

Influence of chelators and iron ions on the production and degradation of H2O2 by beta-amyloid-copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.     

Fabrication of a miniature CMOS-based optical biosensor

This work presents a novel, miniature optical biosensor by immobilizing horseradish peroxidase (HRP) or the HRP/glucose oxidase (GOx) coupled enzyme pair on a CMOS photosensing chip with a detection area of 0.5 mm × 0.5 mm. A highly transparent TEOS/PDMS Ormosil is used to encapsulate and immobilize enzymes on the surface of the photosensor. Interestingly, HRP-catalyzed luminol luminescence can be detected in real time on optical H₂O₂ and glucose biosensors. The minimum reaction volume of the developed optical biosensors is 10 μL. Both optical H₂O₂ and glucose biosensors have an optimal operation temperature and pH of 20–25 °C and pH 8.4, respectively. The linear dynamic range of optical H₂O₂ and glucose biosensors is 0.05–20 mM H₂O₂ and 0.5–20 mM glucose, respectively. The miniature optical glucose biosensor also exhibits good reproducibility with a relative standard deviation of 4.3%. Additionally, ascorbic acid and uric acid, two major interfering substances in the serum during electrochemical analysis, cause only slight interference with the fabricated optical glucose biosensor. In conclusion, the CMOS-photodiode-based optical biosensors proposed herein have many advantages, such as a short detection time, a small sample volume requirement, high reproducibility and wide dynamic range.     

Sulfide oxidation with H2O2 catalyzed by manganese complex of N,N′,N″-tris(2-hydroxypropyl)-1,4,7-triazacyclononane

[MnL](ClO₄)₂ (L = N,N′,N″-tris(2-hydroxypropyl)-1,4,7-triazacyclononane) has been tested for catalyzing sulfide oxidation. In the presence of this complex, ethyl phenyl sulfide, butyl sulfide and phenyl sulfide are completely oxidized to the corresponding sulfoxides and sulfones with H₂O₂ as the oxidant. 2-Chloroethyl phenyl sulfide oxidation yield 2-chloroethyl phenyl sulfone and phenyl vinyl sulfone. In ethyl phenyl sulfide oxidation, effects of complex and H₂O₂ concentration and temperature on the reaction rate have been discussed. Through controlling reaction conditions, ethyl phenyl sulfoxide and ethyl phenyl sulfone may be produced selectively. The UV–Vis and electron paramagnetic resonance (EPR) studies on catalyst solution indicate that metal centre of the complex is transformed from Mn(II) to Mn(IV) after the addition of H₂O₂. At 25 °C, rate constant for ethyl phenyl sulfide oxidation is 4.38 × 10⁻³ min⁻¹.     

Nitrosylmyoglobin as antioxidant--kinetics and proposed mechanism for reduction of hydroperoxides

Nitrosylmyoglobin (MbFe^IINO), which is believed to have a protective role during ischemia and reperfusion injury, was oxidized by tert-butyl hydroperoxide (t-BuOOH), and by hydrogen peroxide (H₂O₂) to the nitrite anion and metmyoglobin (MbFe^III). Further characterization of the reaction of MbFe^IINO with excess of t-BuOOH was investigated with respect to reaction stoichiometry, temperature and pH dependence. It was found that the reaction between MbFe^IINO with excess of t-BuOOH followed a simple stoichiometry and had moderate pH and temperature dependence with the activation parameters ΔH^‡ = 57.4 ± 1.4 kJ mol^- 1 and ΔS^‡ = - 112.0 ± 5.1 J mol^- 1 K^- 1, which is consistent with an associative reaction mechanism. Moreover, t-BuOOH-induced oxidation of MbFe^IINO did not result in any detectable formation of the hypervalent myoglobin (Mb) species, i.e. perferrylmyoglobin, (^√MbFe^IV = O) or ferrylmyoglobin (MbFe^IV = O), and hereby differed from H₂O₂-induced oxidation of MbFe^IINO, which results in the formation of MbFe^IV = O. Based on the obtained results and on published data, different mechanisms for the reaction of the MbFe^IINO with t-BuOOH and H₂O₂ are proposed.     

Structures and magnetism of rubidium and cesium salts of dimeric oxovanadium(IV) tartrate(4−) complexes

Complexes of type A₄[VO(tart)]₂·nH₂O, where A = Rb or Cs and tart =d,l-tartrate(4−) (n = 2) or d,d-tartrate(4−) (n = 2 for Rb and n = 3 for Cs), were prepared from an aqueous mixture of V₂O₅, AOH and H₄tart. These complexes were studied by single-crystal X-ray diffraction methods: Rb₄[VO(d,l-tart)]₂·2H₂O, space group P1 with a = 8.156(1),b = 8.246(1),c = 8.719(1)Å, = 66.09(1)°, β = 65.07(1)°, γ = 82.40(1)°,Z = 2, 1917 observed reflections, and final R_w = 0.035; Cs₄[VO(d,l-tart)]₂·2H₂O, space group P₂₁/c with a = 9.350(1),b = 13.728(2),c = 8.479(1)Å, β = 106.77(1)°,Z = 4, 2235 observed reflections, and final R_w = 0.054; Rb₄[VO(d,d-tart)]₂·2H₂O, space group P₄₁₂₂ with a = 8.072(1),c = 32.006(3)Å,Z = 8, 1014 observed reflections and final R_w = 0.038; Cs₄[VO(d,d-tart)]₂·3H₂O, space group P₁₂₂ with a = 8.184(1),c = 33.680(5)Å,Z = 8, 1310 observed reflections, and final R_w = 0.063. Bulk magnetic susceptibility data (1.5–300 K) for these compounds and A₄[VOl,l-tart)]₂·nH₂O (A = Rb, Cs) were obtained on polycrystalline samples. These data were analyzed in terms of a Van Vleck exchange coupled S = 1/2 model which was modified to include an interdimer exchange parameters Θ. Analysis of the low-temperature (1.5–20 K) susceptibility data gave 2J = +1.30 cm⁻¹ and Θ = −1.86 K for Rb₄[VO(d,l-tart)]₂·2H₂O, 2J = +1.16 cm⁻¹ and Θ = −1.69 K for Cs₄[VO(d,l-tart)]₂·2H₂O, 2J = +1.90 cm⁻¹ and Θ = −0.82 K for Rb₄[VO(d,d-tart)]₂·2H₂O, 2J = +2.04 cm⁻¹ and Θ = −0.80 K for Rb₄[VO(l,l-tart)]₂·2H₂O, 2J = +1.52 cm⁻¹ and Θ = −0.25 K for Cs₄[VO(d,d-tart)]₂·3H₂O, and 2J = +1.64 cm⁻¹ and Θ = −0.31 K for Cs₄[VO(l,l-tart)]₂·3H₂O. These results suggest the magnitudes of intradimer (ferromagnetic and interdimer (antiferromagnetic) exchange interactions are similar in these complexes, as observed for the analogous Na salts.