Complexities in horseradish peroxidase-catalyzed oxidation of dihydroxyphenoxazine derivatives: appropriate ranges for pH values and hydrogen peroxide concentrations in quantitative analysis

The highly sensitive, convenient fluorescence assay, based on the oxidation of nonfluorescent 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to highly fluorescent resorufin, is becoming increasingly popular for hydrogen peroxide quantitation. Yet, the intricacies of the horseradish peroxidase-catalyzed oxidation of the reductant substrate Amplex Red by hydrogen peroxide and the resulting resorufin could complicate the assay design and data interpretation. In particular, substrate inhibition and enzyme inactivation at higher hydrogen peroxide concentrations were known to affect the enzyme kinetics and end-point fluorescence. In addition, here we report the spontaneous transformation of resorufin to less or nonfluorescent product(s) in the absence of hydrogen peroxide and horseradish peroxidase. This spontaneous decay of resorufin fluorescence is most prominent in the pH range 6.2-7.7, likely due to general base-catalyzed de-N-acetylation and polymerization of resorufin. From a practical point of view, precautions for properly designing assays for hydrogen peroxide or characterizing hydrogen peroxide-generating systems are discussed based on the spontaneous transformation of resorufin to less fluorescent compound(s), substrate inhibition and enzyme inactivation at higher (>100 microM) hydrogen peroxide concentrations, and enzymatic oxidation of resorufin to nonfluorescent resazurin.     

The cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide. Steady state kinetic mechanism

Initial velocities for the cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide have been measured as functions of both the ferrocytochrome c (0.27-104 microM) and hydrogen peroxide (0.25-200 microM) concentrations at 25 degrees C, 0.01 M ionic strength, and pH 7 in a cacodylate/KNO3 buffer system Eadie-Hofstee plots of the initial velocity as a function of ferrocytochrome c concentration at constant hydrogen peroxide are nonlinear. A mechanism is proposed which includes random addition of the two substrates to the enzyme and a single catalytically active cytochrome c binding site. The mechanism is consistent with prior studies on cytochrome c peroxidase and fits the steady state kinetic data well.     

Kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase]

A kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase within the pH range of 3.7-9.0 has been carried out. It was shown that the reaction of o-dianisidine peroxidase oxidation obeys the Michaelis--Menten kinetics; the kcat and Km values within the pH range used were determined. The optimum of peroxidase catalytic activity during o-dianisidine oxidation was observed at pH 5.0-6.0. The kinetic pattern of the reaction is discussed. It was demonstrated that deprotonation of the group at pK 6.5 decreases the kcat value 60 times. At pH greater than 8.0 an additional ionogenic group controls the enzyme activity.     

Urinary 1-hydroxypyrene as a biomarker of exposure to polycyclic aromatic hydrocarbons: biological monitoring strategies and methodology for determining biological exposure indices for various work environments

This article reviews the published studies on urinary 1-hydroxypyrene (1-OHP) as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) in work environments. Sampling and analysis strategies as well as a methodology for determining biological exposure indices (BEIs) of 1-OHP in urine for different work environments are proposed for the biological monitoring of occupational exposure to PAHs. Owing to the kinetics of absorption of pyrene by different exposure routes and excretion of 1-OHP in urine, in general, 1-OHP urinary excretion levels increase during the course of a workday, reaching maximum values 3-9 h after the end of work. When the contribution of dermal exposure is important, post-shift 1-OHP excretion can however be lower than pre-shift levels in the case where a worker has been exposed occupationally to PAHs on the day prior to sampling. In addition, 1-OHP excretion levels in either pre-shift, post-shift or evening samples increase during the course of a work-week, levelling off after three consecutive days of work. Consequently, ideally, for a first characterization of a work environment and for an indication of the major exposure route, considering a 5-day work-week (Monday to Friday), the best sampling strategy would be to collect all micturitions over 24 h starting on Monday morning. Alternatively, collection of pre-shift, post-shift and evening urine samples on the first day of the work-week and at the end of the work-week is recommended. For routine monitoring, pre-shift samples on Monday and post-shift samples on Friday should be collected when pulmonary exposure is the main route of exposure. On the other hand, pre-shift samples on Monday and Friday should be collected when the contribution of skin uptake is important. The difference between beginning and end of work-week excretion will give an indication of the average exposure over the workweek. Pre-shift samples on the first day of the work-week will indicate background values, and, hence, reflect general environment exposure and body burden of pyrene and/or its metabolites. On the other hand, since PAH profile can vary substantially in different work sites, a single BEI cannot apply to all workplaces. A simple equation was therefore developed to establish BEIs for workers exposed to PAHs in different work environments by using a BEI already established for a given work environment and by introducing a correction factor corresponding to the ratio of the airborne concentration of the sum of benzo(a)pyrene (BaP) equivalent to that of pyrene. The sum of BaP equivalent concentrations represents the sum of carcinogenic PAH concentrations expressed as BaP using toxic equivalent factors. Based on a previously estimated BEI of 2.3 μmol 1-OHP mol^-1 creatinine for coke-oven workers, BEIs of 4.4, 8.0 and 9.8 μmol 1-OHP mol^-1 creatinine were respectively calculated for vertical pin Söderberg workers, anode workers and pre-bake workers of aluminium plants and a BEI of 1.2 μmol 1-OHP mol^-1 creatinine was estimated for iron foundry workers. This approach will allow the potential risk of cancer in individuals occupationally exposed to PAHs to be assessed better.     

The role of peroxide in haem degradation. A study of the oxidation of ferrihaems by hydrogen peroxide.

The oxidation of ferrihaems by H2O2 was studied as a model for haem catabolism. Rates of ferrihaem oxidation were evaluated by using a new computer-based method that measures the loss in catalytic activity of the ferrihaem during oxidation. For protoferrihaem, deuteroferrihaem, coproferrihaem and mesoferrihaem, oxidation proceeded via the monomeric species and no dimer contribution was detectable. The pH-dependence of oxidation was studied in the range 6.5--11. Within experimental error, the data were compatible with an inverse linear dependence on [H+]. This was interpreted in terms of attack by HO2- on monomeric ferrihaem. The specific second-order rate constants for oxidation of monomeric species by HO2- were of the same order of magnitude for all the ferrihaems, and were in the sequence coproferrihaem greater than protoferrihaem greater than mesoferrihaem congruent to deuteroferrihaem. A model is suggested involving formation of a ferrihaem monomerperoxide complex, which may either dissociate with the formation of a peroxidatic intermediate or be involved in an intramolecular oxidation of the ferrihaem. Haem catabolism may occur via the same or a similar intermediate.     

Synthesis of peroxynitrite using isoamyl nitrite and hydrogen peroxide in a homogeneous solvent system

A method for the synthesis of peroxynitrite is described. It involves nitrosation of H2O2 at pH> or = 12.5 by isoamyl or butyl nitrite in mixed solvents of isopropyl alcohol (IPA) and water at 25+/-1 degrees C. Maximum yields of peroxynitrite are obtained after 15 min of incubation at IPA concentrations of 30-70% (v/v). The solutions of peroxynitrite are processed for removal of IPA and isoamyl alcohol by solvent extraction. Unreacted H2O2 is removed by catalytic decomposition on granular MnO(2). The post processed solutions of peroxynitrite are useful in several chemical and biochemical investigations where bolus additions are required. The method as reported is amenable for large scale synthesis as it involves sequential mixing of solvents (water and IPA) to alkali followed by the addition of H2O2 and alkyl nitrite.     

Assessment of occupational exposure to PAHs in an Estonian coke oven plant- correlation of total external exposure to internal dose measured as 1-hydroxypyrene concentration

The exposure of cokery workers to polynuclear aromatic hydrocarbons at an Estonian oil shale processing plant was assessed by using occupational hygiene and biomonitoring measurements which were carried out twice, in midwinter and in the autumn. To assess the external dose of polynuclear aromatic hydrocarbons, pyrene and benzo[a]pyrene concentrations were measured from the breathing zone of workers during a workshift. Skin contamination with pyrene and benzo[a]pyrene was assessed by skin wipe sampling before and after the workshift. As a biomarker of overall exposure to polynuclear aromatic hydrocarbons, and as an integral of all absorption routes of pyrene, 1-hydroxypyrene concentration was measured from post shift urine samples. Of the personal air samples, 18% exceeded the Finnish threshold limit value of benzo[a]pyrene (10 μg m^-3). Mean value (two separate measurements together) for benzo[a]pyrene was 5.7 μg m^-3 and for pyrene, 8.1 μg m^-3. Based on skin wipe sample analyses, the skin contamination was also obvious. The mean value of benzo[a]pyrene in the samples collected after the shift was 1.2 ng cm^-2. Benzo[a]pyrene was not found in control samples. The mean value of urinary 1-hydroxypyrene concentration was 6.0 μmol mol^-1 creatinine for the exposed workers and 0.5 μmol mol^-1 creatinine for the controls. This study undoubtedly shows the usefulness of 1-hydroxypyrene as an indicator of internal dose of polynuclear aromatic hydrocarbons. It can be concluded that the cokery workers at the Kohtla-Järve plant are exposed to high concentrations of polynuclear aromatic compounds, and the exposure level is considerably higher during the winter measurements.     

Validation of the boronate sensor ContPY1 as a specific probe for fluorescent detection of hydrogen peroxide in plants

Studying the implication of hydrogen peroxide in biological processes in plants remains a challenge due to the current shortcomings of H₂O₂-responsive probes. The use of ContPY1, a new fluorescent probe, which is highly selective and sensitive for H₂O₂, was investigated. To validate the use of ContPY1 on plants, we have generated protocols employing cells suspensions and leaves, and measured specifically H₂O₂ production by plants using spectrofluorometry and microscopy.     

Peroxidase catalyzed phenolic compounds oxidation in presence of surfactant Dynol 604: A kinetic investigation

The kinetics of fungal peroxidase-catalyzed phenolic compounds (PCs) oxidation was investigated in presence of acetylenic-based surfactant Dynol 604 at pH 5.5 and 25 °C. It was shown that the presence of ppm concentrations of surfactant did not influence initial rate of PCs oxidation. The calculated apparent bimolecular rate constants were (1.8 ± 0.2) × 10⁵ M⁻¹ s⁻¹, (1.4 ± 0.4) × 10⁷ M⁻¹ s⁻¹, (1.30 ± 0.06) × 10⁷ M⁻¹ s⁻¹ and 1.1 × 10⁸ M⁻¹ s⁻¹ for phenol, 1-naphthol, 2-naphthol and 1-hydroxypyrene, respectively.During an extensive substrates conversion Dynol 604 showed diverse action for different PCs. The oxidation of phenol practically did not change, whereas the surfactant enhanced the conversion of 1- and 2-naphthol and 1-hydroxypyrene in dose response manner. The results accounted by a scheme, which contains a stadium of enzyme inhibition by oligomeric PC oxidation products. The action of the surfactant was explained by avoidance the enzyme active center clothing with the oligomers. The results acquired demonstrate a remarkable increase of substrates conversion in the presence of Dynol 604.     

Methionine regulates copper/hydrogen peroxide oxidation products of Abeta.

Metal-catalysed oxidation (MCO) may play a causative role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta peptide (Abeta), the major biomarker of AD, in the presence of copper ions reduces Cu(2+) to Cu(+) and catalyses the formation of H(2)O(2) that subsequently induces radicals through Fenton chemistry. Abeta is also subject to attack by free radicals, where the presence of Cu(2+) in conjunction with H(2)O(2) catalyses oxygenation, primarily at the methionine sulfur atom. This work investigates MCO of Abeta, to gain further insight into the role of oxidative stress in AD. By combining a fluorescence assay with gel electrophoresis to monitor MCO reactions of Abeta (1-28) in the presence and absence of methionine it was determined that methionine can both protect some residues against MCO and promote the oxidation of Tyr(10) specifically. Electrospray ionization mass spectrometric analysis of methionine MCO products indicated the formation of methionine sulfoxide, methionine sulfone and related hydroxylated products. Similar products could be formed from the oxidation of Met(35) of Abeta and may relate to changes in properties of the peptide following MCO.     

Methionine oxidation by peroxymonocarbonate, a reactive oxygen species formed from CO2/bicarbonate and hydrogen peroxide

Kinetic and thermodynamic evidence is reported for the role of the peroxymonocarbonate ion, HCO4-, as a reactive oxygen species in biology. Peroxymonocarbonate results from the equilibrium reaction of hydrogen peroxide with bicarbonate via the perhydration of CO2. The kinetic parameters for HCO4- oxidation of free methionine have been obtained (k1 = 0.48 +/- 0.08 M(-1)s(-1) by a spectrophotometric initial rate method). At the physiological concentration of bicarbonate in blood ( approximately 25 mM), it is estimated that peroxymonocarbonate formed in equilibrium with hydrogen peroxide will oxidize methionine approximately 2-fold more rapidly than plasma H2O2 itself. As an example of methionine oxidation in proteins, the bicarbonate-catalyzed hydrogen peroxide oxidation of alpha1-proteinase inhibitor (alpha1-PI) has been investigated via its inhibitory effect on porcine pancreatic elastase activity. The second-order rate constant for HCO4- oxidation of alpha1-PI (0.36 +/- 0.06 M(-1)s(-1)) is comparable to that of free methionine, suggesting that methionine oxidation is occurring. Further evidence for methionine oxidation, specifically involving Met358 and Met351 of the alpha1-PI reactive center loop, has been obtained through amino acid analyses and mass spectroscopic analyses of proteolytic digests of the oxidized alpha1-PI. These results strongly suggest that HCO4- should be considered a reactive oxygen species in aerobic metabolism.     

Determination of polycyclic aromatic hydrocarbons in ambient particulate matter PM2.5 and 1-hydroxypyrene in children from an area near an oil refinery in Northeast Mexico

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with carcinogenic and mutagenic effects. Urban areas characteristically have high concentrations of PAHs associated with vehicle traffic and industry; among these oil refining stands out. One of the six refineries in Mexico has operated in the Metropolitan Area of Monterrey since 1979. The objective of this study was to evaluate exposure to PAHs by determining 1-hydroxypyrene (1-OHP) concentration in children living in an urban area near an oil refinery as well as to determine the relative health risk by exposure to PAHs in this population. This study included 90 children of both sexes aged 5–12 years. Two urine samples were collected, one in summer and one in winter to determine urinary 1-OHP. At the same time, the concentrations of PAHs associated with PM_2.5 were collected in the study area. The mean concentration of 1-OHP was 0.14 μmol/mol of creatinine. Twelve PAHs were quantified, seven are potential carcinogenics and one (benzo[a]pyrene) is carcinogenic for humans, and used as an indicator of cancer relative risk from exposure to PAHs. The annual average concentration of benzo[a]pyrene suggest an excess lifetime cancer risk of 1/100,000 habitants, in the study area.     

Copper, zinc superoxide dismutase plus hydrogen peroxide: a catalytic system for human lipoprotein oxidation

We report for the first time that bovine or human CuZnSOD plus H2O2 can catalyze human lipoprotein oxidation, inducing like free copper ions a typical oxidative kinetics with lag and propagation phases. Free copper released from CuZnSOD by H2O2, but not enzyme peroxidase activity and carbonate radical anion, is responsible for lipoprotein oxidation, which is indeed totally inhibited by copper chelators and BHT but unaffected by bicarbonate. Moreover, lipoprotein oxidation is significantly counteracted by the OH* scavengers formate and azide, which can enter the active site of CuZnSOD and decrease copper release through scavenging of copper-bound OH*; benzoate and ethanol, which cannot enter, are instead ineffective, indicating no oxidative involvement of free OH* escaped from the enzyme active site. The possibility of CuZnSOD/H2O2-catalyzed lipoprotein oxidation in vivo is discussed.     

Model for the exceptional reactivity of peroxiredoxins 2 and 3 with hydrogen peroxide: a kinetic and computational study

Peroxiredoxins (Prx) are thiol peroxidases that exhibit exceptionally high reactivity toward peroxides, but the chemical basis for this is not well understood. We present strong experimental evidence that two highly conserved arginine residues play a vital role in this activity of human Prx2 and Prx3. Point mutation of either ArgI or ArgII (in Prx3 Arg-123 and Arg-146, which are ∼3–4 Å or ∼6–7 Å away from the active site peroxidative cysteine (C_p), respectively) in each case resulted in a 5 orders of magnitude loss in reactivity. A further 2 orders of magnitude decrease in the second-order rate constant was observed for the double arginine mutants of both isoforms, suggesting a cooperative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure suggest strong catalytic effects of H-bond-donating functional groups to the C_p sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cation in the calculations to mimic the functional group of arginine, we were able to locate two transition structures that indicate rate enhancements consistent with our experimentally observed rate constants. Our results provide strong evidence for a vital role of ArgI in activating the peroxide that also involves H-bonding to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H₂O₂ and may have wider implications for protein thiol reactivity toward peroxides.     

Comparison of the urinary excretion time courses of pyrene-1,6-dione,pyrene-1,8-dione and 1-hydroxypyrene in rats intravenously exposed to pyrene

Abstract

The urinary excretion time courses of pyrene-1,6-dione (P16D), pyrene-1,8-dione (P18D) and 1-hydroxypyrene (1-OHP) were compared in Sprague–Dawley and Wistar rats. Groups of five male rats, of about 200 g of body weight, were injected intravenously with 0.05, 0.5, 5 and 50 µmol pyrene kg^?1 of body weight. Urine was collected at 2, 4, 6, 8, 10, 12, 18, 24, 30, 42 and 48 h post-dosing. Pyrene metabolites were measured by high-performance liquid chromatography (HPLC)/fluorescence after enzymatic hydrolysis of the glucurono- and sulfo-conjugates, extraction on Sep-Pak C18 cartridges and, for the analysis of dione metabolites, derivatization to stable diacetoxypyrene molecules. Over the 48-h sampling period, on average 17.4–25.6% of the injected pyrene was excreted overall as P16D, 6.4–8.8% as P18D and 0.6–0.8% as 1-OHP in the urine of Sprague–Dawley rats. By comparison, on average 10.3–14.7% of the intravenous pyrene dose was recovered as P16D, 4.8–6.4% as P18D and 0.3–0.4% as 1-OHP in the urine of Wistar rats. In both strains of rats there was no clear effect of the dose on the 0–48-h cumulative urinary excretion of P18D and 1-OHP over the entire dose range, while the percentage of dose recovered overall as P16D in urine at the highest dose (50 µmol kg^?1) was statistically lower than at the other doses. The 0–48-h cumulative percentage of pyrene dose excreted as metabolites in the urine of Sprague–Dawley rats was also significantly higher than in Wistar rats (p<0.01) exposed under identical conditions. As for the urinary excretion-time courses of the different metabolites, for a given dose and strain of rats, excretion curves of P16D, P18D and 1-OHP generally evolved in parallel. There was also no clear effect of the dose on the excretion rate, thus half-life, of pyrene metabolites, except for P16D in Sprague–Dawley rats at the highest dose where elimination tended to be slower compared with the other doses (p<0.01). The average first-order elimination half-life of P16D, P18D and 1-OHP was 4.0, 5.7 and 4.1 h, respectively, in Sprague–Dawley rats, and 5.1, 6.1 and 5.1 h, respectively, in Wistar rats (all doses combined but excluding the highest dose for P16D). This study showed the relative importance of metabolic pathways leading to diones compared with 1-OHP. These dioxygenated metabolites appear to be interesting biomarkers of pyrene exposure at environmentally and occupationally relevant doses. Their adequacy as biomarkers of human exposure has yet to be confirmed.     

Mechanistic studies on oxidation of hydrogen peroxide and hydrazine by a metal-bound superoxide

In aqueous acetate buffer, hydrogen peroxide and hydrazine reduce the bridging superoxide in [(en)(dien)Co^III(O₂)Co^III(en)(dien)](ClO₄)₅ (1) to the corresponding hydroperoxo complex [(en)(dien)Co^III(μ-O₂H)Co^III(en)(dien)]⁵⁺ (2). In the presence of excess [H₂O₂] and [N₂H₅⁺] over [1], both the reactions obeyed first-order kinetics and exhibited inverse proton dependence. Protonation of 1 at equilibrium generates [(en)(dien)Co^III(μ-O₂H)Co^III(en)(dien)]⁶⁺ (1H), the conjugate acid from 1, which appears to be a kinetic dead-end and that accounts for the observed inverse proton dependence on rate. Reaction rates significantly decrease with increasing proportion of D₂O replacing H₂O in the solvent and an H-atom transfer (HAT) from the reducing species to the bridging superoxide in 1 seems reasonable at the rate step.     

血红素加氧酶-1在对抗过氧化氢引起的血管低反应性中的作用

目的：研究HO-1的诱导剂是否可对抗H2O2引起的血管低反应性,并探讨其作用机制。方法：采用血管环灌流装置,观察胸主动脉环的收缩效应。结果：①SD大鼠腹腔注射高铁血红素后,主动脉HO-1活性和血中CO含量增高;同时,H2O2引起的血管收缩功能下降的现象明显改善。②KATP通道阻断剂优降糖,而非GC抑制亚甲蓝,可取消高铁血红素的抗H2O2损伤的作用。③Hemin＋H2O2组与单纯H2O2组的钙收缩曲线无明显差异。④无钙液中,高铁血红素可抑制H2O2引起的咖啡因和PE诱导的收缩幅度的下降。结论：诱导主动脉HO-1活性增加,可对抗氧化应激引起的血管收缩反应的低下,其机制可能是通过激活KATP通道,影响细胞内贮存钙的释放起作用。而与GC信号转导通路无关。