Effective DNA cleavage by bleomycin-vanadium(IV) complex plus hydrogen peroxide

It has been firstly found that the bleomycin-vanadyl(IV) complex is effectively capable of cleaving DNA in the presence of hydrogen peroxide. The 1:1 bleomycin-VO(IV) complex has been characterized by ESR and electronic absorption spectra, and its ESR parameters (go = 1.982 and Ao = 93.5 G) are indicative of VO(N5) coordination type for the metal-binding environment. The mode of nucleotide sequence cleavage induced by the present bleomycin-VO(IV)-H2O2 complex system was appreciably different from the corresponding Fe(III) complex system. Of special interest is the fact that the bleomycin-vanadium complex system more preferentially attacked G-A(5'----3') sequences than the bleomycin-iron complex system.     

Copper(II) ethylenediaminetetraacetate complex does activate hydrogen peroxide in the presence of biological reductants.

The reaction of CuII (edta) (edta: ethylenediaminetetraacetic acid) with hydrogen peroxide (H2O2) was studied in the pH range of 6.0 to 8.0. CuII (edta) did not react with H2O2 in the all pH range examined in the absence of biological reductants. CuII (edta), however, could react with H2O2 in the presence of biological reductants such as ascorbic acid, cysteine and NADH to give thibarbituric acid (TBA) reactive substance, regardless of the pH. From these results, it is concluded that CuII (edta) cannot be bound to H2O2 and that the change of the redox potential of Cu2+ ion on ligating with edta may cause CuII (edta) to be unable to oxidize H2O2.     

Kinetics of hydrogen peroxide decomposition by catalase: hydroxylic solvent effects

The effect of water–alcohol (methanol, ethanol, propan-1-ol, propan-2-ol, ethane-1,2-diol and propane-1,2,3-triol) binary mixtures on the kinetics of hydrogen peroxide decomposition in the presence of bovine liver catalase is investigated. In all solvents, the activity of catalase is smaller than in water. The results are discussed on the basis of a simple kinetic model. The kinetic constants for product formation through enzyme–substrate complex decomposition and for inactivation of catalase are estimated. The organic solvents are characterized by several physical properties: dielectric constant (D), hydrophobicity (log P), concentration of hydroxyl groups ([OH]), polarizability (α), Kamlet-Taft parameter (β) and Kosower parameter (Z). The relationships between the initial rate, kinetic constants and medium properties are analyzed by linear and multiple linear regression.     

Strong emission increase of a dicarboxyterpyridene europium (III) complex in the presence of citrate and hydrogen peroxide

A substituted 6,6″-dicarboxy-2,2′:6′,2″-terpyridene europium (III) complex (1) responds with an emission wavelength shift and intensity increase to the presence of citrate and hydrogen peroxide, while many oxoanions show no effect.     

Reaction pathways in the decomposition of hydrogen peroxide catalyzed by copper(II)

The kinetics of the decomposition of H(2)O(2) catalyzed by Cu(II) has been studied by the initial-rate method in aqueous phosphate media at near physiological pH. The activity of the catalyst is increased by [Fe(CN)(6)](3-) and decreased by VO(3)(-), CrO(4)(2-) and Zn(II). Three reaction pathways are involved in the Cu(II)-H(2)O(2) reaction, the kinetic orders of the catalyst being 1 (rate constant k1), 2 (rate constant k2) and 3 (rate constant k3). The three pathways present fractional apparent orders (>1) in H(2)O(2) and base catalysis. The apparent activation energies associated to rate constants k1, k2 and k3 are 102+/-4, 65+/-8 and 61+/-5 kJ mol(-1). Free-radical chain mechanisms are proposed for the three pathways.     

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.     

Metal-catalyzed oxidation of alpha-synuclein in the presence of Copper(II) and hydrogen peroxide

alpha-Synuclein is a component of abnormal protein depositions of Lewy bodies and senile plaques found in Parkinson's and Alzheimer's diseases, respectively. By using chemical coupling reagents such as dicyclohexylcarbodiimide or N-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline, the protein was shown to experience self-oligomerization in the presence of either copper(II) or Abeta25-35. The oligomers which appeared as a ladder on a 10-20% Tricine/SDS-PAGE have been suggested to participate in the formation of protein aggregations by possibly providing a nucleation center. Since oxidatively modified protein could increase its own tendency toward protein aggregation, metal-catalyzed oxidation of alpha-synuclein has been examined with copper(II) and hydrogen peroxide in the absence of the coupling reagent. Intriguingly, the protein was also self-oligomerized into an SDS-resistant ladder on the gel. This biochemically specific copper-mediated oxidative oligomerization was shown to be dependent upon the acidic C-terminus of alpha-synuclein because the C-terminally truncated proteins such as alpha-syn114 and alpha-syn97 were not affected by the metal and hydrogen peroxide. More importantly, the oxidative oligomerization was synergistically enhanced by the presence of Abeta25-35, indicating that the peptide interaction with alpha-synuclein facilitated the copper(II) binding to the acidic C-terminus and subsequent oxidative crosslinking. It has been, therefore, suggested that abnormalities in copper and H(2)O(2) homeostasis and certain pathological factors functionally similar to the Abeta25-35 could play critical roles in the metal-catalyzed oxidative oligomerization of alpha-synuclein, which may lead to possible protein aggregation and neurodegenerations.     

Activity of magnetite-immobilized catalase in hydrogen peroxide decomposition

The present work analyzes the activity in decomposition of H₂O₂ using magnetite-immobilized catalase. The support of catalase is a glutaraldehyde-treated magnetite (Fe₃O₄). The data obtained in the H₂O₂ decomposition are analyzed. The fitting of the initial rate of the H₂O₂ decomposition versus hydrogen peroxide concentration data is discussed using a specific program for enzyme kinetics modeling (Leonora). The free catalase from Aspergillus niger (3.5 or 10 U/mL) does not show substrate inactivation up to 0.4 M H₂O₂. The immobilized catalase at low catalyst concentration shows substrate inhibition. Using 1 mg/mL of supported catalase the predicted maximum activity is higher than in the case of the free catalase at similar catalase concentration, although the optimum temperature is lower (40 °C versus 60 °C).     

Kinetic studies on the reaction of ethylenediaminetetraacetatochromium(III) complex with hydrogen peroxide

The rate of reaction of [Cr(III)Y]_aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO₃)] at various temperatures. The general rate equation, Rate = $\text{k}{}_1\text{+}\text{k}{}_2\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}\text{1 +}\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}$ [Cr(III)Y]_aq[H₂O₂] holds over the pH range 5–9. The decomposition reaction of H₂O₂ is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k₁ and k₂ were found to be 1.75 × 10^?2 M^?1 s^?1 and 0.174 M^?1 s^?1 at 303 K respectively, where k₂ is the rate constant for the aquo species and k₂ is that for the hydroxo complex. The respective activation enthalpies (ΔH^*₁ = 58.9 and ΔH^*₂ = 66.5 KJ mol^?1) and activation entropies (ΔS^*₁ = ?85 and ΔS^*₂ = ?40 J mol^?1 deg^?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK₁, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).     

The reaction of ferrous leghemoglobin with hydrogen peroxide to form leghemoglobin(IV)

Ferrous leghemoglobin reacts with hydrogen peroxide to form the stable product, leghemoglobin(IV). The reaction follows second order kinetics (k = 2.24 X 10(4) M-1 S-1 at 20 degrees C) and may be regarded as a single-step, two-electron oxidation. Ferric leghemoglobin is not an intermediate. The oxidation state of leghemoglobin(IV) is established by reductive titration with dithionite; 2 eq of dithionite are required to convert 1 mol of leghemoglobin(IV) to ferrous leghemoglobin. An outstanding property of leghemoglobin(IV) is its stability, little change is noted after 12 h at 25 degrees C. Leghemoglobin(IV) differs from the higher oxidation states of other hemoglobins and myoglobins in that it does not react with hydrogen peroxide to form the oxygenated protein.     

Mechanism of DNA cleavage induced by sodium chromate(VI) in the presence of hydrogen peroxide

Reactivities of chromium compounds with DNA were investigated by the DNA sequencing technique using 32P 5'-end-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Incubation of double-stranded DNA with sodium chromate(VI) plus hydrogen peroxide or potassium tetraperoxochromate(V) led to the cleavage at the position of every base, particularly of guanine. Even without piperidine, the formation of oligonucleotides was observed, suggesting the breakage of the deoxyribose-phosphate backbone. ESR studies using hydroxyl radical traps demonstrated that hydroxyl radical is generated both during the reaction of sodium chromate(VI) with hydrogen peroxide and the decomposition of potassium tetraperoxochromate(V), and that hydroxyl radical reacts significantly not only with mononucleotides but also with deoxyribose 5-phosphate. ESR studies using a singlet oxygen trap demonstrated that singlet oxygen is also generated both by the same reaction and decomposition, and reacts significantly with deoxyguanylate, but scarcely reacts with other mononucleotides. Furthermore, ESR studies suggested that tetraperoxochromate(V) is formed by the reaction of sodium chromate(VI) with hydrogen peroxide. These results indicate that sodium chromate(VI) reacts with hydrogen peroxide to form tetraperoxochromate(V), leading to the production of the hydroxyl radical, which causes every base alteration and deoxyribose-phosphate backbone breakage. In addition, sodium chromate(VI) plus hydrogen peroxide generates singlet oxygen, which subsequently oxidizes the guanine residue. The mechanism by which both hydroxyl radical and singlet oxygen are generated during the reaction of sodium chromate(VI) with hydrogen peroxide was presented. Finally, the possibility that this reaction may be one of the primary reactions of carcinogenesis induced by chromate(VI) is discussed.     

Oxidative deamination by hydrogen peroxide in the presence of metals

Various amines, including lysine residue of bovine serum albumin, were oxidatively deaminated to form the corresponding aldehydes by a H 2 O 2 /Cu 2+ oxidation system at physiological pH and temperature. The resulting aldehydes were measured by high-performance liquid chromatography. We investigated the effects of metal ions, pH, inhibitors, and O 2 on the oxidative deamination of benzylamine by H 2 O 2 . The formation of benzaldehyde was the greatest with Cu 2+ , and catalysis occurred with Co 2+ , VO 2+ , and Fe 3+ . The reaction was greatly accelerated as the pH value rose and was markedly inhibited by EDTA and catalase. Dimethyl sulfoxide and thiourea, which are hydroxyl radical scavengers, were also effective in inhibiting the generation of benzaldehyde, indicating that the reaction is a hydroxyl radical-mediated reaction. Superoxide dismutase greatly stimulated the reaction, probably due to the formation of hydroxyl radicals. O 2 was not required in the oxidation, and instead slightly inhibited the reaction. We also examined several oxidation systems. Ascorbic acid/O 2 /Cu 2+ and hemoglobin/H 2 O 2 systems also converted benzylamine to benzaldehyde. The proposed mechanism of the oxidative deamination by H 2 O 2 /Cu 2+ system is discussed.     

Reconstitution of the water-oxidizing complex in manganese-depleted photosystem II preparations using synthetic binuclear Mn(II) and Mn(IV) complexes: production of hydrogen peroxide

Reconstitution of Mn-depleted PSII particles with synthetic binuclear Mn complexes (one Mn(II)₂ complex and one Mn(IV)₂ complex) was examined. In both cases the electron-transfer rates in the reconstituted systems were found to be up to 75–82% of that measured in native PSII but the oxygen evolution activity remained lower (<5–40%). However, hydrogen peroxide was also produced by the reconstituted samples. These samples therefore represent a new type of reconstituted PSII that generates hydrogen peroxide as the final product in reconstituted PSII centers.     

Accelerated CuZn-SOD-mediated oxidation and reduction in the presence of hydrogen peroxide

Copper, zinc-superoxide dismutase (CuZn-SOD) is a cytosolic, antioxidant enzyme that scavenges potentially damaging superoxide radical (()O(2)(-)). Under the proper conditions, CuZn-SOD also catalyzes the oxidation and reduction of certain small molecules. Here, we demonstrate that increased exposure to hydrogen peroxide (H(2)O(2)), a by-product of the ()O(2)(-) scavenging reaction, dramatically increases the ability of CuZn-SOD to oxidize melatonin and reduce S-nitrosoglutathione (GSNO). After a 15min in vitro incubation with CuZn-SOD and 1mM H(2)O(2), 76% of the melatonin was oxidized, compared to 52% with 0.25mM H(2)O(2), and just 9% without H(2)O(2). Pre-incubation with 1mM H(2)O(2) resulted in a 100% increase in the rate of GSNO breakdown by CuZn-SOD in the presence of glutathione (GSH) compared to untreated CuZn-SOD. Collectively, these data suggest that even small increases in intracellular H(2)O(2) levels may result in the oxidation and/or reduction of small molecules critical for proper cellular function.     

Kinetics of formation of the primary compound (compound I) from hydrogen peroxide and turnip peroxidases.

A kinetic study of the reaction of two turnip peroxidases (P1 and P7) with hydrogen peroxide to form the primary oxidized compound (compound I) has been carried out over the pH range from 2.4 to 10.8. In the neutral and acidic pH regions, the rates depend linearly on hydrogen peroxide concentration whereas at alkaline pH values the rates display saturation kinetics. A compound is made with the cyanide binding reaction to peroxidases since the two reactions are influenced in the same manner by ionization of groups on the native enzymes. Two different ionization processes of peroxidase P1 with pKa values of 3.9 and 10 are required to explain the rate pH profile for the reaction with H2O2. Protonation of the former group and ionization of the latter causes a decrease in the rate of reaction of the enzyme with H2O2. In the case of peroxidase P7 a minimum model involves three ionizable groups with pKa values of 2.5, 4 and 9. Protonation of the former two groups and ionization of the latter lowers the reaction rate. In the pH-independent region, the rate of formation of compound I was measured as a function of temperature. From the Arhenius plots the activation energy for the reaction was calculated to be 2.9 +/- 0.1 kcal/mol for P1 and 5.4 +/- 0.3 kcal/mol for P7. However, the rates are independent of viscosity in glycerol-water mixtures up to 30% glycerol.     

Products of Cu(II)-catalyzed oxidation of the N-terminal fragments of alpha-synuclein in the presence of hydrogen peroxide

Reactive oxygen species (ROS) may provide the covalent modifications of amino acid residues in proteins, formation of protein-protein cross-linkages, and oxidation of the protein backbone resulting in protein fragmentation. In an attempt to elucidate the products of the copper(II)-catalyzed oxidation of the (1-17), (1-28), (1-39) and (1-39)(A30P) fragments of alpha-synuclein, the high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods and Cu(II) /hydrogen peroxide as a model oxidizing system were employed. Peptide solution (0.50 mM) was incubated at 37 degrees C for 24 h with metal:peptide:hydrogen peroxide molar ratio 1:1:4 in phosphate buffer, pH 7.4. Oxidation targets for all peptide studied are the methionine residues (M(1), M(5)). Incubation 24 h of the (1-28), (1-39) and (1-39)(A30P) fragments in aerobic conditions lead to the oxidation of one methionine residue to methionine sulfoxide. Reaction of hydrogen peroxide with all fragments of alpha-synuclein resulted in oxidation of two methionine residues (M(1), M(5)) to methionine sulfoxides. For the Cu(II):peptide:hydrogen peroxide 1:1:4 molar ratio systems the further oxidation of methionine residues to sulfone was observed. The cleavage of the peptide bond M(1)-D(2) for all peptides studied was observed as metal binding residues. For the (1-39) and (1-39)(A30P) fragments of alpha-synuclein the molecular ions with lower molecular masses (A(11)-Y(39), E(13)-Y(39)) were also detected.