Presence Of Excess Hydrogen Peroxide Research Articles

Formation and transformation of nitroso compounds of ketoxime isomers in ketone ammoximation catalyzed by hollow titanium silicalite

2-Nitrosopropane as the isomer of acetoxime was first found in ammoxidation of acetone with hydrogen peroxide catalyzed by hollow titanium silicalite (HTS). Experimental investigation indicates that the formation of 2-nitrosopropane was closely related to the pore size and surface acidity of HTS zeolite and significantly affected by reaction conditions, and further revealed that once formed, this isomer could be easily converted into main product acetoxime and also turned into by-product 2-nitropropane in the presence of excess hydrogen peroxide or O2. Based on the experimental results in combination with the analysis of reaction mechanism, it was suggested that the pathways of acetone ammoxidation catalyzed by HTS could involve both hydroxylamino intermediate and imino intermediate. The former was dominant in the production of acetone oxime, while the latter was mainly responsible for the formation of 2-nitropropane. In the latter case, an acetone first reacted with ammonia to form an imine and then interacted with an active center of Ti-OOH on catalyst to generate an unstable 2-nitrosopropane.

Improved rate of substrate oxidation catalyzed by genetically-engineered myoglobin

This study showcases the potential of unnatural amino acids to enable non-natural functions when incorporated in the protein scaffold of heme metalloproteins. For this purpose, a genetically-engineered myoglobin (Mb) mutant was created by incorporating redox-active 3-amino-l-tyrosine (NH2Tyr) into its active site, replacing the distal histidine (H64) with NH2Tyr. In peroxide-shunt assays, this variant exhibits an increased rate of turnover for thioanisole and benzaldehyde oxidation as compared to the wild-type (WT) Mb. Indeed, in the presence of excess hydrogen peroxide (H2O2), a 9-fold and 81-fold increase in activity was observed over multiple turnovers for thioanisole sulfoxidation and benzoic acid formation, respectively. The increased oxidation activity in the H64NH2Tyr Mb mutant underlined the role of NH2Tyr in the distal active-site scaffold in peroxide activation. Kinetic, electrochemical, and EPR spectroscopic experiments were performed. On the basis of these studies, it is argued that the single NH2Tyr residue within the Mb variant simultaneously serves the role of the conserved His/Arg-pair within the distal pocket of horseradish peroxidase.

Dissection of the radical reactions linked to fetal hemoglobin reveals enhanced pseudoperoxidase activity.

In the presence of excess hydrogen peroxide (H2O2), ferrous (Fe+2) human hemoglobin (Hb) (α2β2) undergoes a rapid conversion to a higher oxidation ferryl state (Fe+4) which rapidly autoreduces back to the ferric form (Fe+3) as H2O2 is consumed in the reaction. In the presence of additional H2O2 the ferric state can form both ferryl Hb and an associated protein radical in a pseudoperoxidative cycle that results in the loss of radicals and heme degradation. We examined whether adult HbA (β2α2) exhibits a different pseudoenzymatic activity than fetal Hb (γ2α2) due to the switch of γ to β subunits. Rapid mixing of the ferric forms of both proteins with excess H2O2 resulted in biphasic kinetic time courses that can be assigned to γ/β and α, respectively. Although there was a 1.5 fold increase in the fast reacting γ /β subunits the slower reacting phases (attributed to α subunits of both proteins) were essentially the same. However, the rate constant for the auto-reduction of ferryl back to ferric for both proteins was found to be 76% higher for HbF than HbA and in the presence of the mild reducing agent, ascorbate there was a 3-fold higher reduction rate in ferryl HbF as opposed to ferryl HbA. Using quantitative mass spectrometry in the presence of H2O2 we found oxidized γ/β Cys93, to be more abundantly present in HbA than HbF, whereas higher levels of nitrated β Tyr35 containing peptides were found in HbA samples treated with nitrite. The extraordinary stability of HbF reported here may explain the evolutionary advantage this protein may confer onto co-inherited hemoglobinopathies and can also be utilized in the engineering of oxidatively stable Hb-based oxygen carriers.

Fluorescent derivatization of aromatic carboxylic acids with horseradish peroxidase in the presence of excess hydrogen peroxide.

The fluorescent derivatization of aromatic carboxylic acids by the catalytic activity of horseradish peroxidase (HRP) in the presence of excess H2O2 was investigated. Four monocarboxylic acids, nine dicarboxylic acids, and two tricarboxylic acids, all of which are non- or weakly fluorescent, were effectively converted into fluorescent compounds using this new method. This technique was further developed for the fluorometric determination of trace amounts of terephthalic acid (3c) and lutidinic acid (2b), and linear calibration curves for concentrations between 2.5 and 20.0 nmol of terephthalic acid (3c) and 1.0 and 10.0 nmol of lutidinic acid (2b) were demonstrated. Compound III, an intermediate of HRP, played an essential role in this process. Additionally, lactoperoxidase and manganese peroxidase, peroxidases similar to HRP, showed successful fluorescent derivatization of nicotinic acid (1b), lutidinic acid (2b), and hemimellitic acid (4a) in the presence of excess H2O2.

Fate of thiabendazole through the treatment of a simulated agro-food industrial effluent by combined MBR/Fenton processes at μg/L scale

This study has been carried out to assess the performance of a combined system consisting of a membrane bioreactor (MBR) followed by an advanced oxidation process (Fenton/Photo-Fenton) for removing the fungicide thiabendazole (TBZ) in a simulated agro-food industrial wastewater. Previous studies have shown the presence of TBZ in the effluent of an agro-food industry treated by activated sludge in a sequencing batch reactor (SBR), thus reinforcing the need for alternative treatments for removal. In this study, a simulated agro-food industry effluent was enriched with 100 μg L−1 TBZ and treated by combined MBR/Fenton and MBR/solar photo-Fenton systems. Samples were directly injected into a highly sensitive liquid chromatography-triple quadrupole-linear ion trap-mass spectrometer (LC-QqLiT-MS/MS) analytical system to monitor the degradation of TBZ even at low concentration levels (ng L−1). Results showed that the biological treatment applied was not effective in TBZ degradation, which remained almost unaltered; although most dissolved organic matter was biodegraded effectively. Fenton and solar photo-Fenton, were assayed as tertiary treatments. The experiments were run without any pH adjustment by using an iron dosage strategy in the presence of excess hydrogen peroxide. Both treatments resulted in a total degradation of TBZ, obtaining more than 99% removal in both cases. To assure the total elimination of contaminants in the treated waters, transformation products (TPs) of TBZ generated during Fenton degradation experiments were identified and monitored by liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS). Up to four TPs could be identified. Two of them corresponded to mono-hydroxylated derivatives, typically generated under hydroxyl radicals driven processes. The other two corresponded with the hydrolysis of the TBZ molecule to yield benzoimidazole and thiazole-4-carboxamidine. All of them were also degraded during the treatment.

Fluorescent Derivatization of Xanthurenic Acid and Nicotinic Acid with Horseradish Peroxidase in the Presence of Excess Hydrogen Peroxide

The fluorescent derivatization of tryptophan metabolites (xanthurenic acid, nicotinic acid, picolinic acid, and 3-hydroxyanthranilic acid) by the catalytic activity of horseradish peroxidase (HRP) was investigated in the presence of excess H(2)O(2). Non-fluorescent xanthurenic acid (XA) and nicotinic acid (NA) were converted into a fluorescent compound with maximum excitation and emission wavelengths at 325 and 425 nm, and 318 and 380 nm, respectively. This fluorescent derivatization was developed for the fluorometric determination of trace amounts of XA and NA. The calibration curves were linear from 1.0 to 10.0 nmol XA and from 5.0 to 20.0 nmol NA in a 1.0-mL sample solution. The UV spectra of the reaction solutions suggested that compound III as an intermediate of HRP played an essential role in this fluorescent derivatization with HRP.

The effect of oxidative stress on the mutation rate of Mycobacterium tuberculosis with impaired catalase/peroxidase function

To determine the effect of oxidative stress on isoniazid-resistant Mycobacterium tuberculosis deficient in catalase/peroxidase activity to varying degrees through mutation in katG. The mutation rate was determined for a set of isogenic strains with different katG alleles giving different catalase and/or peroxidase activities following exposure to the oxidizing agent, hydrogen peroxide. Mutants were selected on rifampicin, and the location and nature of the mutation were identified by sequencing the rpoB gene. No evidence was found to suggest that strains that had impaired catalase/peroxidase activity were hypermutable, and the presence of excess hydrogen peroxide had no effect on the mutation rate. An unusual pattern of mutations in rpoB was observed in catalase-deficient strains with only 3 of 66 having mutations within the rifampicin resistance-determining region. The mutation rate of M. tuberculosis in response to oxidative stress is not increased in strains with significant deficits in catalase and peroxidase activity. Our data suggest that isoniazid-resistant strains compensate for their reduced ability to detoxify oxidative stress effectively. Interestingly, mutations were found in unusual locations at positions similar to those found in clinical isoniazid-resistant strains.

The crystal structure of peroxymyoglobin generated through cryoradiolytic reduction of myoglobin compound III during data collection

Myoglobin has the ability to react with hydrogen peroxide, generating high-valent complexes similar to peroxidases (compounds I and II), and in the presence of excess hydrogen peroxide a third intermediate, compound III, with an oxymyoglobin-type structure is generated from compound II. The compound III is, however, easily one-electron reduced to peroxymyoglobin by synchrotron radiation during crystallographic data collection. We have generated and solved the 1.30 A (1 A=0.1 nm) resolution crystal structure of the peroxymyoglobin intermediate, which is isoelectric to compound 0 and has a Fe-O distance of 1.8 A and O-O bond of 1.3 A in accordance with a Fe(II)-O-O- (or Fe(III)-O-O2-) structure. The generation of the peroxy intermediate through reduction of compound III by X-rays shows the importance of using single-crystal microspectrophotometry when doing crystallography on metalloproteins. After having collected crystallographic data on a peroxy-generated myoglobin crystal, we were able (by a short annealing) to break the O-O bond leading to formation of compound II. These results indicate that the cryoradiolytic-generated peroxymyoglobin is biologically relevant through its conversion into compound II upon heating. Additionally, we have observed that the Xe1 site is occupied by a water molecule, which might be the leaving group in the compound II to compound III reaction.

Novel sol-gel immobilization of horseradish peroxidase employing a detergentless micro-emulsion system

A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted ofn-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above 95% of its initial activity for 48h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized using the novel sol-gel method described here was more stable than enzymes immobilized using the other method.

Removal of aqueous phenol by Arthromyces ramosus peroxidase

The removal of aqueous phenol by Arthromyces ramosus peroxidase (ARP), in the presence of high molecular weight polyethylene glycol (PEG) as a protective additive, was investigated to study (i) the stoichiometry between phenol removal and peroxide consumption, (ii) enzyme inactivation in the presence of excess hydrogen peroxide, and (iii) the effect of reactor operating mode on phenol removal. The stoichiometry was observed to increase with increasing phenol conversion, and reached a maximum value of 0.9 mol phenol/mol peroxide when nearly complete phenol conversion was achieved. Spectral scans indicated that P-670 did not form with ARP in the presence of high concentrations of peroxide and in the absence of a reducing substrate. As long as phenol concentration was not limiting, the turnover capacity varied by approximately 3% of its average value of 76 400. This indicates that only marginal improvement in the efficiency of enzyme use would be achieved by stepped addition of enzyme. Similarly, sequential addition of hydrogen peroxide over an extended period of time yielded only a slight improvement in phenol removal from the reaction mixture. Key words: Arthromyces ramosus peroxidase, enzyme inactivation, phenol removal, reactor operation, stoichiometry.

The role of redox-active amino acids on compound I stability, substrate oxidation, and protein cross-linking in yeast cytochrome C peroxidase.

The role of two tryptophans (Trp51 and Trp191) and six tyrosines (Tyr36, Tyr39, Tyr42, Tyr187, Tyr229, and Tyr236) in yeast cytochrome c peroxidase (CcP) has been probed by site-directed mutagenesis. A series of sequential mutations of these redox-active amino acid residues to the corresponding, less oxidizable residues in lignin peroxidase (LiP) resulted in an increasingly more stable compound I, with rate constants for compound I decay decreasing from 57 s(-1) for CcP(MI, W191F) to 7 s(-1) for CcP(MI, W191F,W51F,Y187F,Y229F,Y236F,Y36F,Y39E,Y42F). These results provide experimental support for the proposal that the stability of compound I depends on the number of endogenous oxidizable amino acids in proteins. The higher stability of compound I in the variant proteins also makes it possible to observe its visible absorption spectroscopic features more clearly. The effects of the mutations on oxidation of ferrocytochrome c and 2,6-dimethoxyphenol were also examined. Since the first mutation in the series involved the change of Trp191, a residue that plays a critical role in the electron transfer pathway between CcP and cyt c, the ability to oxidize cyt c was negligible for all mutant proteins. On the other hand, the W191F mutation had little effect on the proteins' ability to oxidize 2,6-dimethoxyphenol. Instead, the W51F mutation resulted in the largest increase in the k(cat)/K(M), from 2.1 x 10(2) to 5.0 x 10(3) M(-1) s(-1), yielding an efficiency that is comparable to that of manganese peroxidase (MnP). The effect in W51F mutation can be attributed to the residue's influence on the stability and thus reactivity of the ferryl oxygen of compound II, whose substrate oxidation is the rate-determining step in the reaction mechanism. Finally, out of all mutant proteins in this study, only the variant containing the Y36F, Y39E, and Y42F mutations was found to prevent covalent protein cross-links in the presence of excess hydrogen peroxide and in the absence of exogenous reductants. This finding marks the first time a CcP variant is incapable of forming protein cross-links and confirms that one of the three tyrosines must be involved in the protein cross-linking.

Assessment of the Potential of Arthromyces Ramosus Peroxidase to Remove Phenol from Industrial Wastewaters

The ability of Arthromyces ramosus peroxidase (ARP) to catalyze phenol removal from aqueous solution was investigated under a number of conditions both in the presence and in the absence of a protective additive, high molecular weight polyethylene glycol. The stoichiometry of phenol removal to peroxide consumption was observed to increase from 1.0 to 1.2 as initial phenol, peroxide and enzyme concentrations were increased. ARP was found to exhibit thermal stability comparable to that of HRP, but considerably less than that of SBP when incubated at pH 7.0 at temperatures between 35 and 55 °C. The activation energy was estimated to be 69 kJ mol−1. The benefit of using PEG to slow the rate of the permanent inactivation of ARP was demonstrated. Approximately 13.7 times less enzyme was required for a given phenol removal in the presence of excess PEG than was needed in its absence. ARP was found to be as efficient as SBP, but less efficient than HRP, at removing phenol at 25 °C and pH 7.0. Modeling suggested that the enzyme undergoes rapid and essentially irreversible inactivation in the presence of excess hydrogen peroxide. Spectral observation indicated that the inactive form of ARP produced in the presence of hydrogen peroxide was not solely compound III (a temporary inactivation), nor was it verdohaemoprotein (a permanent inactivation), as is the case when HRP undergoes inactivation by hydrogen peroxide.

Assay of Peracid in the Presence of Excess Hydrogen Peroxide

ABSTRACT A simple and rapid colorimetric method for the determination of peracids in the presence of excess hydrogen peroxide is described. The method is based on the selective destruction of hydrogen peroxide by the enzyme catalase, a procedure which leaves the peracid unaffected. The residual solution, then containing only peracid, can be assayed using the stoichiometric oxidation of 2, 2′-azino-bis-(3-ethyl-benzthiazoline-6-sulfonate) diammonium salt (ABTS) in the presence of peroxidase, a reaction that can be quantified photometrically at 405 run. The double-enzyme method was extended to microtiter plate-based systems, thus providing an easy way of analyzing multiple samples for their peracid content.

Oxidation of ochratoxin A by an Fe-porphyrin system: model for enzymatic activation and DNA cleavage.

Ochratoxin A (OTA, 1) is a fungal toxin that facilitates single-strand DNA cleavage, DNA adduction, and lipid peroxidation when metabolically activated. To model the enzymatic activation of OTA, we have employed the water-soluble iron(III) meso-tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS) oxidation system. In its presence, OTA has been found to facilitate single-strand cleavage of supercoiled plasmid DNA through production of reactive oxygen species (ROS) (i.e., the hydroxyl radical, HO(*)). The reaction of OTA with the FeTPPS oxidation system also generated three hydroxylated products (chlorine atom still attached), which was taken as evidence for production of the known hydroxylated metabolites (2-4) of OTA. This result suggested that the FeTPPS system served as a reasonable model for the enzymatic activation of OTA. When the reaction of OTA with FeTPPS was carried out in the presence of excess hydrogen peroxide (H(2)O(2)) and sodium ascorbate, a hydroquinone species (OTHQ, 5) was detected in which an OH group has replaced the chlorine atom of OTA. The production of OTHQ (5) was dependent on the presence of the reducing agent, sodium ascorbate, which suggested that the oxidation catalyst furnished the quinone derivative OTQ (6) that was subsequently reduced to OTHQ (5) by ascorbate. Utilizing a synthetic sample of OTHQ (5), the hydroquinone was found to undergo autoxidation with a t(1/2) of 11.1 h at pH 7.4, and to possess a pK(a) value of 8.03 for the phenolic oxygen ortho to the carbonyl groups. Our findings imply that the hydroquinone (OTHQ) and quinone (OTQ) metabolites of OTA have the ability to cause alkylation/redox damage and have allowed us to propose a viable pathway for oxidative damage by OTA.

Kinetics of the Formation of p-670 and of the Decay of Compound III of Horseradish Peroxidase

The kinetics of the decay of compound III of horseradish peroxidase formed from compound II and excess hydrogen peroxide and of the formation of p-670 from this preparation were studied as a function of pH at 25°C and constant ionic strength of 0.11. Compound III undergoes an irreversible formation of p-670 and a slow, spontaneous decay to ferriperoxidase via compound II and the rate of compound III decay is less than that of p-670 formation at all pH values. Both reactions follow pseudo first order kinetics at all pH values. The observed rate constant for compound III decay decreases with increase in pH with a minimum at pH 8.0. Also the observed rate constant for p-670 formation increases with increase in pH with a maximum also at pH 8.0. The slow decay of our preparation of compound III and its unique formation of p-670 as compared to oxyperoxidase prepared from ferrous horseradish peroxidase and molecular oxygen (J. B. Wittenberg, R. W. Noble, B. A. Wittenberg, E. Antonini, M. Brunori, and J. Wyman (1967)J. Biol. Chem.242, 625–634) are discussed in terms of the presence of excess hydrogen peroxide in our preparation. Ferriperoxidase and p-670 are formed from compound III in two parallel reactions.

Kinetic modeling of the epoxidation of natural rubber with in‐situ–formed peracid

AbstractIn the presence of excess hydrogen peroxide and formic acid the latex stage epoxidation of natural rubber is pseudo–first order in nature. A kinetic model is developed to predict the maximum extent of epoxidation for the reaction system and its variation with increasing acid concentration. Th dependence of the overall rate constant on acid concentration is also studied.

Peroxo-oxometallate formation under phase transfer conditions

The peroxidation of molybdenum(VI) and tungsten(VI) was investigated in the biphasic system water-chlorobenzene to elucidate the nature of the entities formed and transferred into the organic phase by phase transfer catalysts (PTC). It was observed that application of excess hydrogen peroxide to the acidic aqueous phase resulted in the exhaustive disaggregation of isopolyoxometallates, and the simple diperoxo-metallates were transferred by PTC. When the heteroatom (X, in the form of PO 4 3−or AsO 4 3−) was applied in a quantity commensurable with that of the metal ion (M), heteropolyoxometallates could be extracted in which the ratio M/X was determined by the concentrations of X and PTC. In the presence of excess hydrogen peroxide, peroxoheteropolyoxomolybdates with an O act/M ratio of 2:1 were formed. Observations suggested that in the latter species the XO 4 or X 2O 7 core is coordinated by diperoxo-oxometallate units. Through the use of different PTCs, several peroxoheteropolyoxometallates were isolated and investigated.

Oxidation of Dimethylsulphoxide to Formaldehyde by Oxyhaemoglobin and Oxyleghaemoglobin in the Presence of Hydrogen Peroxide is Not Mediated by “Free” Hydroxyl Radicals

In the presence of excess hydrogen peroxide, human oxyhaemoglobin and oxyleghaemoglobin from soybean root nodules cause oxidation of dimethylsulphoxide to formaldehyde. This reaction is inhibited by thiourea but not by phenylalanine, HEPES, mannitol or arginine. It is concluded that dimethylsulphoxide oxidation is not mediated by "free" hydroxyl radicals, consistent with previous conclusions that intact haemoglobin, leghaemoglobin or myoglobin molecules do not react with H2O2 to form hydroxyl radicals detectable outside the protein.

Spectrophotometric determination of iodide and iodine at parts-per-million level as a thiocyanate complex

A simple method is described for the determination of iodide or iodine in aqueous solutions by the generation and photometric measurement of an iodine thiocyanate complex, I(SCN)$sub 2$$sup -$, in the presence of excess hydrogen peroxide. Because other halides do not produce serious interferences when present in excesses of several thousandfold, separation from these anions can be avoided in many natural samples. The transition metal ions interfered to varying extents. It is suggested that pretreatment of natural samples with a cation exchange resin could remove interfering cations to acceptable levels. (JGB)

The analysis of combustion products : The effect of hydrogen peroxide on the reaction of potassium permanganate with organic peroxides in acid solution

Although methyl and ethyl hydroperoxides alone do not react with potassium permanganate in acid solution at room temperature, they can be oxidised by this reagent in the presence of hydrogen peroxide. The apparent equivalents (as compared with hydrogen peroxide) are much lower than usual but, in the presence of excess hydrogen peroxide, the titre due to the organic peroxide is proportional to its original concentration. This forms the basis of a simple titration method which, though empirical, is both rapid and accurate. Since t-butyl hydroperoxide does not react in this way, it may be possible to differentiate the higher peroxides from lower peroxides.