Determination of urinary oxalate with alkylamine glass-bound sorghum oxalate oxidase and horseradish peroxidase

Oxalate in urine was analyzed using sorghum oxalate oxidase and horseradish peroxidase immobilized on alkylamine glass through glutaraldehyde. The minimum detection limit was 0.46 g/0.1 ml urine. The recovery of added oxalate was 97.5%. Within and between assay coefficients of variation were <3.5% and <6.5% respectively. A good correlation (r=0.9234) was found between oxalate values obtained by a commercial kit method and the present method. The method is unaffected by Cl– and NO3– found in urine.     

Determination of hydrogen peroxide generated by reduced nicotinamide adenine dinucleotide oxidase

Hydrogen peroxide can be conveniently determined using horseradish peroxidase (HRP) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). However, interference occurs among assay components in the presence of reduced nicotinamide adenine dinucleotide (NADH) that is also a substrate of NADH oxidase. So, depletion of NADH is required before using the HRP method. Here, we report simple and rapid procedures to accurately determine hydrogen peroxide generated by NADH oxidase. All procedures developed were based on the extreme acid lability of NADH and the stability of hydrogen peroxide, because NADH was decomposed at pH 2.0 or 3.0 for 10 min, while hydrogen peroxide was stable at pH 2.0 or 3.0 for at least 60 min. Acidification and neutralization were carried out by adjusting sample containing NADH up to 30 microM to pH 2.0 for 10 min before neutralizing it back to pH 7.0. Then, hydrogen peroxide in the sample was measured using the HRP method and its determination limit was found to be about 0.3 microM. Alternatively, hydrogen peroxide in samples containing NADH up to 100 microM could be quantitated using a modified HRP method that required an acidification step only, which was found to have a determination limit of about 3 microM hydrogen peroxide in original samples.     

Transient formation of the oxo–iron(IV) porphyrin radical cation during the reaction of iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)porphyrin with hydrogen peroxide in aqueous solution

The reaction of iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)porphyrin (Fe(III)TMPyP) with hydrogen peroxide (H₂O₂) and the catalytic activity of the reaction intermediates on the luminescent peroxidation of luminol in aqueous solution were studied by using a double‐mixing stopped‐flow system. The observed luminescence intensities showed biphasic decay depending on the conditions. The initial flashlight decayed within <1 s followed by a sustained emission for more than 30 s. Computer deconvolution of the time‐resolved absorption spectra under the same conditions revealed that the initial flashlight appeared during the formation of the oxo–iron(IV) porphyrin, TMPyPFe(IV) = O, which is responsible for the sustained emission. The absorption spectra 0.0–0.5 s did not reproduce well by a simple combination of the two spectra of Fe(III)TMPyP and TMPyPFe(IV) = O, indicating that transient species was formed at the initial stage. Addition of uric acid (UA) caused a significant delay in the initiation of the luminol emission as well as in the formation of the TMPyPFe(IV) = O. Both of them were completely diminished in the presence of UA equimolar with H₂O₂, while mannitol had no effect at all. The delay of the light emission as well as the appearance of TMPyPFe(IV) = O was directly proportional to the [UA]₀ but other kinetic profiles were not changed significantly. Based on these observations and the kinetic analysis, we confirmed the involvement of the oxo–iron(IV) porphyrin radical cation, (TMPyP)^·+Fe(IV) = O, as an obligatory intermediate in the rate‐determining step of the overall reaction, Fe(III)TMPyP + H₂O₂ → TMPyPFe(IV) = O, with a rate constant of k = 4.3 × 10⁴/mol/L/s. The rate constants for the reaction between the (TMPyP)^·+Fe(IV) = O and luminol, and between the TMPyPFe(IV) = O and luminol were estimated to be 3.6 × 10⁶/mol/L/s and 1.31 × 10⁴/mol/L/s, respectively. Copyright © 2003 John Wiley & Sons, Ltd.     

Occurrence of an oxalate oxidase in Sorghum leaves

An oxalate oxidase found in the 15 000 g supernatant of 10-day-old sorghum leaves exhibited a pH optimum of 5 and a temperature optimum of 45° and was unaffected by Na⁺. The enzyme activity remained linear up to 10 min and the apparent K_m for oxalate was 2.4 × 10^?5 M. The enzyme activity was strongly inhibited by sodium dithionite and α,α′-dipyridyl. Inhibition by the latter was specifically reversed by Fe²⁺. The activity of the dialysed enzyme was restored by the addition of Fe²⁺ and FAD. Inhibition of the enzyme by iodoacetate, p-chloromercuribenzoate and N-methylmaleimide revealed that SH groups at the active site are essential.     

Reaction mechanism of surfactant‐sensitized chemiluminescence of bis(2,4,6‐trichlorophyenyl) oxalate and hydrogen peroxide induced by gold nanoparticles

The chemiluminescence (CL) of bis(2,4,6‐trichlorophyenyl) oxalate with hydrogen peroxide in the present of cationic surfactant and gold nanoparticles was studied. The CL emission was obviously enhanced in the presence of surfactant at a suitable concentration, with a synergetic catalysis effect exhibited. Different sizes of gold nanoparticles (15 and 50 nm) showed different effects on CL intensity. Mechanisms of the CL reaction and sensitization effect are discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Enhanced chemiluminescence in the oxidation of luminol and an isoluminol cortisol conjugate by hydrogen peroxide in reversed micelles

The chemiluminescent oxidation of luminol and an isoluminol cortisol conjugate (ABICOR) by hydrogen peroxide has been studied in cetyltrimethylammonium bromide (CTAB) reversed micelles in octane-chloroform (1 : 1). The maximum chemiluminescence intensity of both compounds is dependent on the initial concentrations of the H₂O₂ and substrates, the pH value of the micelle polar phase and the H₂O/CTAB ratio. The optimum pH ranged from 8.5 to 9.5. Under comparable conditions, the chemiluminescence intensity for luminol was 15-fold higher than for the ABI-COR conjugate. A mechanism of oxidation of the substrates in reversed micelles is proposed and the possible mechanisms of inhibition by the substrate and oxidant is discussed.     

Low-temperature bleaching of cotton induced by glucose oxidase enzymes and hydrogen peroxide activators

Glucose oxidase enzymes were used to produce hydrogen peroxide from glucose and oxygen in aqueous solutions. Different working conditions, that is, temperature, aeration with liquefied air, presence of cotton fibre and time of enzyme activity, were tested in order to obtain a solution with the highest possible concentration of hydrogen peroxide. The hydrogen peroxide produced was transformed into different peracids which could bleach the cotton fabric under mild conditions, at a pH between 7 and 8 and at a temperature of around 60°C. The conversion or activation of hydrogen peroxide was conducted with the bleach activators TAED, NOBS and TBBC. The concentrations of hydrogen peroxide and peracids in the solutions were measured with sodium thiosulphate titrations.

The results indicated that the formation of hydrogen peroxide with glucose oxidase was effective under optimal conditions, which are 50°C, pH 4.6 and aeration. Convenient activators for the conversion of hydrogen peroxide into peracids were TAED and TBBC, which enabled attainment of a relatively high degree of whiteness at pH 7.5 and temperature 50°C. Using the activator NOBS under these conditions did not provide enough peracid to markedly improve whiteness.     

Long-Term Chemiluminescent Signal Is Produced in the Course of Luminol Peroxidation Catalyzed by Peroxidase Isolated from Leaves of African Oil Palm Tree

Optimal conditions were found for the oxidation of luminol by hydrogen peroxide in the presence of peroxidase isolated from leaves of the African oil palm tree Elaeis guineensis (AOPTP). The pH range for maximal chemiluminescence intensity (8.3-8.6) is similar for AOPTP, horseradish, and Arthromyces ramosus peroxidases and slightly different from that for tobacco peroxidase (9.3). Increasing the buffer concentration decreases the chemiluminescence intensity. As in the case of other anionic peroxidases, the catalytic efficiency of AOPTP does not depend on the presence of enhancers (4-iodophenol and 4-hydroxycinnamic acid) in the reaction medium. The detectable limit of AOPTP assayed by luminol peroxidation is 2·10^–12 M. The long-term chemiluminescence signal produced during AOPTP-dependent luminol peroxidation is a characteristic feature of the African oil palm enzyme. This feature in combination with its very high stability suggests that AOPTP will be a promising tool in analytical practice.     

草酸氧化酶基因转化烟草的研究

为研究草酸氧化酶基因(OxO)对植物抗病的作用,将含有CaMV 35s启动子的植物表达载体以根癌农杆菌(Agrobacterium tumefaciens)介导的叶盘方法,转化了烟草97131。具有卡那霉素抗性的再生植株经PCR检测,得到了与阳性对照一致的470bp的片段,进一步对PCR产物测序表明OxO基因已整合进烟草基因组中。在对草酸的耐受性实验中,转基因烟草对草酸的抗性明显高于未转化烟草。     

A novel chemiluminescent flow injection analysis of trace amounts of rutin by its inhibition of the luminol–hydrogen peroxide reaction catalyzed by tetrasulfonated colbalt phthalocyanine

Based on the inhibition effect of rutin on the luminol–hydrogen peroxide chemiluminescence (CL) system catalyzed by tetrasulfonated colbalt phthalocyanine (CoTSPc), a sensitive flow‐injection CL method has been developed for the determination of rutin. The CL reaction mechanism was carefully investigated by examining CL emission spectra, UV–visible spectra and variation of reaction conditions. It was found that there existed a linear relationship between CL intensity and the concentration of rutin in the range of 8.0 × 10^?9 to 1.0 × 10^?6 mol L^?1, and the detection limit is 3.8 × 10^?9 mol L^?1. This proposed method is sensitive, convenient and simple, and has been applied to the determination of rutin in commercial rutin tablets with satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.     

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC’s properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 μg/cm². The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.     

Modulation of bovine serum amine oxidase activity by hydrogen peroxide

Bovine serum amine oxidase (BSAO), reduced by excess amine under limited turnover conditions, was over 80% inactivated by H(2)O(2) upon oxygen exhaustion. The UV-Vis spectrum and the reduced reactivity with carbonyl reagents showed that the cofactor topaquinone (TPQ) was stabilized in reduced form. The protein large M(r) (170 kDa) prevented the identification of modified residues by amino acid analyses. Minor changes of the Cu(2+) EPR signal and the formation of a radical at g = 2.001, with intensity a few percent of that of the Cu(2+) signal, unaffected by a temperature increase, suggest that Cu(2+)-bound histidines were not oxidized and the radical was not the Cu(+)-semiquinolamine in equilibrium with Cu(2+)-aminoquinol. It may derive from the modification of a conserved residue in proximity of the active site, possibly the tyrosine at hydrogen-bonding distance of TPQ C-4 ionized hydroxyl. The inactivation reaction appears to be a general feature of copper-containing amine oxidases. It may be part of an autoregulatory process in vivo, possibly relevant to cell adhesion and redox signaling.     

Kinetic simulation studies on the transient formation of the oxo‐iron(IV) porphyrin radical cation during the reaction of iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)‐porphyrin with hydrogen peroxide in aqueous solution

High‐valent oxo‐iron(IV) species are commonly proposed as the key intermediates in the catalytic mechanisms of iron enzymes. Water‐soluble iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)porphyrin (Fe(III)TMPyP) has been used as a model of heme‐enzyme to catalyse the hydrogen peroxide (H₂O₂) oxidation of various organic compounds. However, the mechanism of the reaction of Fe(III)TMPyP with H₂O₂ has not been fully established. In this study, we have explored the kinetic simulation of the reaction of Fe(III)TMPyP with H₂O₂ and of the catalytic reactivity of FeTMPyP in the luminescent peroxidation of luminol. According to the mechanism that has been established in this work, Fe(III)TMPyP is oxidized by H₂O₂ to produce (TMPyP)^·+Fe(IV)=O (k1 = 4.5 × 10⁴/mol/L/s) as a precursor of TMPyPFe(IV)=O. The intermediate, (TMPyP)^·+Fe(IV)=O, represented nearly 2% of Fe(III)TMPyP but it does not accumulate in suf?cient concentration to be detected because its decay rate is too fast. Kinetic simulations showed that the proposed scheme is capable of reproducing the observed time courses of FeTMPyP in various oxidation states and the decay pro?les of the luminol chemiluminescence. It also shows that (TMPyP)^·+Fe(IV)=O is 100 times more reactive than TMPyPFe(IV)=O in most of the reactions. These two species are responsible for the initial sharp and the sustained luminol emissions, respectively. Copyright © 2003 John Wiley & Sons, Ltd.     

Determination of serum oxalate using peroxyoxalate chemiluminescence of free oxalic acid

We describe a new sensitive and specific method for determination of oxalate in human serum. By using the chemiluminescence decay of monoperoxyoxalic acid very low concentrations of oxalate (200 nmol/L) can be determined. The mean serum oxalate level in apparently healthy controls was 14.5 ± 8.5 m?mol/L. Supplementation of ascorbic acid leads to an increase in serum oxalate level. While serum oxalate concentrations of calcium oxalate stone formers (x = 16.4 ± 9.8 m?mol/L) are not significantly different from the control group, an extreme increase of serum oxalate is evident in haemodialysis patients. The serum oxalate concentration decreased during dialysis treatment from 141.4 ± 32.1 m?mol/L to 36.4 ± 12.7 m?mol/L.     

Hybridization of horseradish peroxidase-labeled probes and detection by enhanced chemiluminescence

This article describes the use of probes directly labeled with horseradish peroxidase in conjunction with enhanced chemiluminescence, which allows a flexible approach to hybridizations and detections. This system may be used with the following applications: Southern blots, Northern blots, colony and plaque screening for positive clones, YAC clone screening, and PCR products detection. The major steps required for the use of directly labeled HRP probes are hybridization, stringent washes, and detection.     

Cell wall oxalate oxidase modifies the ferulate metabolism in cell walls of wheat shoots

Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide.     

Reactions of copper complexes with oxygen radicals generated by human neutrophils

The intensity of the chemiluminescence of unstimulated human neutrophils in the presence of luminol was used to investigate the effects of low-molecular-weight copper complexes at the cellular level. In different models (superoxide dismutase mimetic activity, inhibition of haematoporphyrin derivative/light-induced lysis of cells), the biological activity of the complexes exceeded the activity of the ligands alone.     

Decolourization of paprika dye effluent with hydrogen peroxide produced by glucose oxidase

Abstract

Hydrogen peroxide was produced from bran by a two-step process using cellulase/xylanase and glucose oxidase, sequentially. The decolourization efficiency of the produced reagent was tested using paprika oil dye (effluent from industrial source) and high levels of colour removal (96%) were achieved after saponification pre-treatment and hydrogen peroxide application. The method is economically and environmentally advantageous since lower energy and chemical input are needed and wastewater pollution is considerably reduced. At the same time, the utilization of waste materials was successfully achieved.     

Reactivation of horseradish peroxidase with imidazole for continuous determination of hydrogen peroxide using a microflow injection–chemiluminescence detection system

A method for reactivation of inactivated horseradish peroxidase (HRP) was studied and exploited in an assay for hydrogen peroxide (H₂O₂). Addition of imidazole into a mobile phase made continuous determination of hydrogen peroxide (H₂O₂) possible by micro?ow injection based on horseradish‐catalysed luminol chemiluminescence. For reproducible determination of H₂O₂ with HRP, the inactivation of HRP via protonation of the active sites of HRP caused by reaction with H₂O₂ must be avoided. We successfully reactivated protonated HRP (inactive HRP) with exogenous imidazole in the mobile phase of the micro?ow injection system. The imidazole successfully removed the attached proton from the inactive sites of the HRP. This assay was reproducible (within‐run reproducibility, CV = 4.0%) and the detection limit for H₂O₂ was 5 pmol. Copyright © 2003 John Wiley & Sons, Ltd.     

Amines for detection of dopamine by generation of hydrogen peroxide and peroxyoxalate chemiluminescence

A range of nitrogen-containing compounds (alkyl amines, piperazines, cyclohexylamines and nitrogen heterocyclics) were investigated for generation of hydrogen peroxide from dopamine and detection by peroxyoxalate chemiluminescence. Imidazole, ethyleneurea and allantoin among the nitrogen heterocyclic compounds tested generated hydrogen peroxide from dopamine following incubation at 60°C, pH 9.5–10.5, for 0–30 min. Imidazole was the most effective for generation of hydrogen peroxide, but imidazole derivatives with a primary amine side chain (histamine) or thiol (ethylenethiourea) were not effective. The presence of a ketone group (ethyleneurea, allantoin) did not hinder the reaction. Under optimal conditions (30 min incubation, 50 mmol/L imidazole) 10.5 nmol of dopamine could be detected. The cyclohexylamines tested produced low amounts of hydrogen peroxide (0.09–2.74% of light intensity with imidazole), and the piperazines and the alkyl amines tested produced no detectable hydrogen peroxide. Imidazole reacts with the phenolic groups of dopamine in a different manner from monoamine oxidase, and a reagent containing imidazole, ethyleneurea or allantoin was useful for non-enzymatic detection of dopamine by peroxyoxalate chemiluminescence.© John Wiley & Sons, Ltd.