Peroxidase-like catalytic activity of Mn- and Fe-tetrakis(4-carboxyphenyl)porphines bound to aminopropyl-glass bead in oxidative reaction of heterocyclic amines

Fe- or Mn-tetrakis(4-carboxyphenyl)porphine (Fe- and Mn-TCPP) bound to aminopropyl-glass bead (Fe- and Mn-TCPP_gs) was examined for the peroxidase (POD)-like function in order to develop a solid catalyst which can exhibit POD-like activity without adsorbing heterocyclic amines (HCAs). Mn-TCPP in aqueous solution had only a slight POD-like catalytic activity on HCAs (IQ and MeIQ). As for Fe-TCPP, it was impossible to examine the POD-like activity since it reacted with hydrogen peroxide in a liquid reaction system. However, both Fe- and Mn-TCPP when immobilized on aminopropyl-glass bead via peptide bond (Fe- and Mn-TCPP_gs), catalyzed the oxidative reaction of mutagenic HCAs with hydrogen peroxide. The catalytic activity of Fe- and Mn-TCPP_gs was investigated in more detail using as a substrate IQ and MeIQ which were oxidized more rapidly among the tested HCAs. Consequently, the optimal conditions for the oxidative reaction catalyzed by Fe- and Mn-TCPP_gs were determined. In addition, ESI-mass and absorption spectra of oxidation products of IQ and MeIQ showed that they are dimers. Thus, it was demonstrated that a solid catalyst with POD-like activity can be obtained by immobilizing Fe- and Mn-TCPPs on aminopropyl-glass beads.     

Determination of hydrogen peroxide by iron(III) complex of thiacalix[4]arenetetrasulfonate on a modified ion-exchanger with peroxidase-like catalytic activity.

An iron(III) complex of thiacalix[4]arenetetrasulfonate on a modified anion-exchanger (Fe3+-TCAS(A-500)) has shown high peroxidase-like activity at pH 5 - 6 for the reaction of quinoid-dye formation between 3-methyl-2-benzothiazolinone hydrazone and N-(3-sulfopropyl)aniline in the presence of hydrogen peroxide. Utilizing the peroxidase-like activity of Fe3+-TCAS(A-500) for this reaction, a method using Fe3+-TCAS(A-500) was applied for the spectrophotometric determination of hydrogen peroxide. The calibration curve by the method using Fe3+-TCAS(A-500) was linear over the range from 1 to 10 microg of hydrogen peroxide in a 1 ml sample solution. The apparent molar absorptivity for hydrogen peroxide was 2.4 x 10(4) l mol(-1) cm(-1). which was about 80% of that by peroxidase under the same conditions. This determination method of hydrogen peroxide using Fe3+-TCAS(A-500) was applied for the determination of glucose in diluted normal and abnormal control serum I and II.     

Determination of hydrogen peroxide with N,N-diethylaniline and 4-aminoantipyrine by use of an anion-exchange resin modified with manganese-tetrakis(sulphophenyl)porphine, as a substitute for peroxidase

Amberlite IRA 900 anion-exchange resin modified with manganese-tetrakis(sulphophenyl)-porphine has been used as a catalyst instead of peroxidase for the determination of hydrogen peroxide by the reaction 2H(2)O(2) + N,N-diethylaniline + 4-aminoantipyrine (catalyst)--> quinonoid dye (lambda(max) 550 nm) + 4H(2)O. The apparent molar absorptivity for hydrogen peroxide was 1.1 x 10(4) 1.mole(-1).cm(-1), coefficient of variation 0.7%. This value is approximately 84% of that obtained by the use of peroxidase as catalyst. Similar conditions to those in the enzymatic reaction were suitable for use of the modified resin as catalyst, and the results show it to be a good substitute for peroxidase in this reaction system.     

Flow-injection determination of L-histidine with an immobilized histidine oxidase from Brevibacillus borstelensis KAIT-B-022 and chemiluminescence detection.

A chemiluminometric flow injection analytical system for the quantitation of L-histidine is described. Histidine oxidase (EC 1.4.3.-) from Brevibacillus borstelensis KAIT-B-022 was immobilized on tresylated poly(vinyl alcohol) beads and packed into a stainless-steel column. The hydrogen peroxide produced was detected chemiluminometrically by a flowthrough sensor containing immobilized peroxidase (EC 1.1 1.1.7). The maximum sample throughput was 10 h(-1). The calibration graph was linear from 0.05 to 5 mM; the detection limit (signal to noise ratio = 3) was 0.01 mM. The activity of immobilized histidine oxidase reduced to 65% of the initial value after 350 injections. The system was applied to the determination of L-histidine in fish meat, such as salmon, tunny, bonito, and mackerel.     

Fluorometric determination of ethanol in liquor samples by flow-injection analysis using an immobilized enzyme-reactor column with packing prepared by coupling alcohol oxidase and peroxidase onto chitosan beads

A flow-injection system was developed for the determination of ethanol with an immobilized enzyme-reactor column. This system, which consisted of hand-made reactor columns packed with alcohol oxidase and horseradish peroxidase immobilized onto chitosan beads, and a fluorometric detector, was applied to the determination of ethanol in liquor samples. Under the recommended conditions, the ethanol, which was present in the pretreated samples, was converted to hydrogen peroxide when it was passed through the immobilized alcohol oxidase (AOD) column with 0.1 mol/dm3 phosphate buffer (pH 7.0). A sample can be analyzed with this system in <10 min. The calibration curve for ethanol was linear from 2.0 to 0.1 mg/dm3. The determination limit, which was defined by the difference between the sample peak and blank peak, was estimated to be 50 microg/dm3 for ethanol. Interferences from some substances present in actual liquor samples decreased the analytical response and activity of the immobilized AOD-reactor column, but they were removed by dilution and pretreatment with an octyldecylsilane cartridge.     

High performance liquid chromatography of glucose using a post-column reactor of immobilized enzyme followed by electrochemical detection (glucose-LCEC)

A sensitive and selective, reasonably fast method for the determination of glucose content has been developed. A glucose oxidase immobilized column was coupled to a small-size anion exchange column/borate buffer chromatograph. The hydrogen peroxide produced in the enzyme reaction was detected directly by an amperometric detector using a platinum working electrode. The detection limit was 0.03 ppm (1.5 x 10(-7) M, 3 pmol/injection). The linear dynamic range was three orders of magnitude at least. The system was stable and reproducible both in short- and long-term operation. The proposed method is suitable for analysis of complicated matrices of biological samples because of its good selectivity and sensitivity.     

Evaluation of luminol-H(2)O(2)-KIO(4) chemiluminescence system and its application to hydrogen peroxide, glucose and ascorbic acid assays

A novel chemiluminescence (CL) system was evaluated for the determination of hydrogen peroxide, glucose and ascorbic acid based on hydrogen peroxide, which has a catalytic-cooxidative effect on the oxidation of luminol by KIO(4). Hydrogen peroxide can be directly determined by luminol-KIO(4)-H(2)O(2) CL system. The detection limit was 3.0x10(-8) mol l(-1) and the calibration graph was linear over the range of 2.0x10(-7)-6.0x10(-4) mol l(-1). The relative standard deviation of H(2)O(2) was 1.1% for 2.0x10(-6) mol l(-1) (N=11). Glucose was indirectly determined through measuring the H(2)O(2) generated by the oxidation of glucose in the presence of glucose oxidase at pH 7.6. The present method provides a source for H(2)O(2), which, in turn, coupled with the luminol-KIO(4)-H(2)O(2) CL reaction system. The CL was linearly correlated with glucose concentration of 0.6-110 mug ml(-1). The relative standard deviation was 2.1% for 10 mug ml(-1) (N=11). Detection limit of glucose was 0.08 mug ml(-1). Ascorbic acid was also indirectly determined by the suppression of luminol-KIO(4)-H(2)O(2) CL system. The calibration curve was linear over the range of 1.0x10(-7)-1.0x10(-5) mol l(-1) of ascorbic acid. The relative standard deviation was 1.0% for 8.0x10(-7) mol l(-1) (N=11). Detection limit of ascorbic acid was 6.0x10(-8) mol l(-1). These proposed methods have been applied to determine glucose, ascorbic acid in tablets and injection.     

Flow-injection determination of glutathione with amperometric monitoring of the enzymatic reaction

Glutathione sulfhydryl oxidase is immobilized on oxirane-acrylic beads (Eupegit-C) and packed in a small column. The simple system for glutathione comprises the immobilized enzyme column and a flow-through membrane-covered platinum/silver/silver chloride electrode pair for detection of hydrogen peroxide. The calibration graph for glutathione was linear from 0.05 to 1.0 mM for 200-μl samples. The assay took 3 min. The relative standard deviation for 0.5 mM glutathione was 2% (n=10).     

Free and Ca-Alginate Beads Immobilized Horseradish Peroxidase for the Removal of Reactive Dyes: an Experimental and Modeling Study

The aim of this work was to remove the dyes Reactive Blue 221 (RB 221) and Reactive Blue 198 (RB 198) of synthetic effluent using the immobilized enzyme horseradish peroxidase (HRP) in Ca-alginate beads. Experimental parameters affecting the dye removal process such as the effect of pH, temperature, hydrogen peroxide concentration, mass capsules, and reuse were evaluated, and a numerical model of mass transfer was developed. A maximum removal of 93 and 75%, respectively, for the dyes RB 221 and RB 198, at pH 5.5 and temperature of 30 °C, concentration of hydrogen peroxide of 43.75 μM for dye RB 221 and 37.5 μM for the dye of RB 198 was obtained. A removal reaction of 180 min for RB 221 and 240 min for RB 198 was observed. Three reuse cycles of use of immobilized enzyme were achieved for both dyes. The numerical model proposed led to a good fit compared to experimental data. The HRP enzyme immobilized in Ca-alginate capsules showed a great potential for biotechnological applications, especially for the removal of reactive dyes.     

Kinetic flow-injection determination of hydrogen peroxide by use of iron(III)-catalyzed coloration and its application to the determination of biological substances.

A kinetic flow-injection (FI) method is described for the determination of hydrogen peroxide. This method is based on an iron(III)-catalyzed oxidative coupling of 4-aminoantipyrine with N,N-dimethylaniline by hydrogen peroxide. By measuring the change in the absorbance of the dye formed at 560 nm, 1 x 10(-6) - 6 x 10(-4) M hydrogen peroxide could be determined with a sampling rate of 15 h(-1). The relative standard deviation (n = 30) was 0.8% for 5 x 10(-5) M hydrogen peroxide. There was little interference of the co-existing ions and compounds. After introducing some immobilized enzyme reactors to the FI system, the proposed method allowed the determination of glucose and uric acid ranging from 1 x 10(-6) to 6 x 10(-4) M with relative standard deviations of below 2%. The applicability of the method was demonstrated by determining these substances in serum samples.     

Oxidation of ferrocene and some substituted ferrocenes in the presence of horseradish peroxidase

The enzyme catalysed oxidation of ferrocene and some substituted ferrocenes to the corresponding ferricinium ions by hydrogen peroxide in the presence of native or immobilized horseradish peroxidase has been studied. Initial and maximum rates of oxidation have been determined. It was found that the oxidation was independent of the hydrogen peroxide concentration. The oxidation of ferrocene was effected also by horseradish peroxidase in a coupled system with glucose oxidase in the absence of any added hydrogen peroxide.     

Liposome-based flow injection enzyme immunoassay for theophylline

Preliminary results are presented on the development of a sensitive, quantitative immunoassay based on a regenerable, flow injection analysis system incorporating a double-amplification approach. The double amplification is achieved by means of liposome-encapsulated peroxidase enzyme molecules which are released subsequent to a competitive immunological reaction with analyte molecules for immobilized antibodies. The released peroxidase enzymatically cleaves, from an organofluorine substrate, fluoride ions which are then potentiometrically measured. The entire process is carried out in a flow injection analysis system. The competition between the analyte molecules (theophylline) and theophylline-derivatized liposomes for immobilized antibody sites in flow-through immunoreactor column results in unbound liposomes being carried downstream where they are ruptured in the presence of hydrogen peroxide andp-fluorophenol. The peroxidase molecules released react enzymatically to produce fluoride ions which are measured with an ion-selective electrode. The immunoreactor column is then regenerated with a chaotropic agent and the next sample or calibration solution is injected. By means of column regeneration and calibration, accurate quantitation can be achieved; a feature missing from conventional batch-type immunoassays. By means of this liposome/enzyme double-amplification approach, theophylline was determined over a range of concentrations from 0.2 to 4000 ng/ml. The detection limit of 200 pg/ml corresponds to about 100 femtomole of theophylline measured in the 100 l sample injected.Dedicated to Professor W. Simon on the occasion of his 60th birthday     

Determination of purine nucleosides and their bases by high-performance liquid chromatography using co-immobilized enzyme reactors

A selective and sensitive assay of inosine, guanosine, hypoxanthine, guanine and xanthine by high-performance liquid chromatography with immobilized enzyme reactors was developed. The separation was achieved on a Capcell Pak C18 column (15 cm x 0.46 cm I.D.) with a mobile phase of 0.1 M phosphate buffer (pH 8.0) containing 7 mM sodium 1-hexanesulphonate and 0.1 mM p-hydroxyphenylacetic acid. The fluorimetric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of these compounds, which were oxidized to yield hydrogen peroxide in the presence of immobilized enzyme (purine nucleoside phosphorylase, guanase and xanthine oxidase). Enzyme reactions occurred sufficiently without post-column addition of reagents. Enzymes that catalysed the conversion of purine compounds were co-immobilized on aminopropyl controlled-pore glass packed in stainless-steel tubing. The detection limits were 30-200 pg per injection.     

Sensitive flow-injection determination of l-lactate in human blood with immobilized enzyme columns and fluorimetric detection

Enzyme columns prepared by packing l-lactate oxidase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series. Hydrogen peroxide formed in the enzymatic conversion of l-lactate in the first column is mixed with 3-(p-hydroxyphenyl)propionic acid before passage through the peroxidase column and fluorimetric measurement. Linear calibration was obtained for 0.5–500 pmol of L-lactate in 20 μl of 1000-fold diluted, deproteinated whole blood. A rapid sampling rate (60 h^?1) was possible.     

Flow-through chemiluminescence sensor using immobilized histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007 and peroxidase for selective determination of histamine.

A flow sensor with immobilized oxidases is proposed for the determination of histamine in fish meat. Chemiluminometric measurement of histamine was based on the luminol reaction with hydrogen peroxide produced by immobilized histamine oxidase (EC 1.4.3.-.) and peroxidase (EC 1.11.1.7.) within a flow cell. Histamine oxidase was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The oxidase and peroxidase were coimmobilized covalently on tresylated hydrophilic vinyl polymer beads and packed into transparent PTFE; the tubing was used as the flow cell. One assay for histamine was done at intervals of 2 min without carryover. The calibration curve for histamine was linear from 0.1 microM to 50 microM. The response was reproducible within 1.25% of the relative standard deviation for 115-replicate injections of 50 microM histamine. The sensor system was applied to the determination of histamine in fish meat extracts.     

One pot glucose detection by [Fe(III)(biuret-amide)] immobilized on mesoporous silica nanoparticles: an efficient HRP mimic

An [Fe(III)(biuret-amide)] complex has been immobilized onto mesoporous silica nanoparticles via Cu(I) catalyzed azide-alkyne click chemistry. This hybrid material functions as an efficient peroxidase mimic and was successfully used for the quantitative determination of hydrogen peroxide and glucose via a one-pot colorimetric assay.     

芯片式流通池顺序注射可更新表面反射光谱法用于酶反应检测

将芯片式流通池顺序注射可更新表面反射光谱法用于酶反应检测。HRP催化H2O2氧化BPR底物的反应用于对H2O2的检测。此反应体系与葡萄糖氧化酶联用，用于对血清中葡萄糖的检测。     

HPLC Determination of Hepatic Cholesterol 7α-Hydroxylase Activity Using Immobilized Cholesterol Oxidase and Chemiluminescence Detection

Abstract

This paper describes an HPLC method for the determination of cholesterol 7α-hydroxylase activity, at high or low activity levels, that is sensitive and specific for 7α-hydroxycholesterol. The method relies on the generation of hydrogen peroxide by oxidation of 7α-hydroxycholesterol using the enzyme cholesterol oxidase which has been immobilized on porous glass beads. The hydrogen peroxide is subsequently detected by chemiluminescence generated by reaction of peroxide with bis-(2, 4, 6-trichlorophenyl)-oxalate (TCPO), a commonly used chemiluminescence reagent specific for peroxides. In the procedure, sample preparation is limited to extraction of the incubation mixture and injection of the concentrated extract.     

Detection of Oxidase Generated Hydrogenperoxide by a Solid State Peroxyoxalate Chemiluminescence Detector

Abstract

The compatability of a solid state peroxyoxalate chemiluminescence detector for hydrogen peroxide with an immobilized oxidase reactor is investigated. As a model system glucose oxidase immobilized by electrostatic forces on an ion-exchanger or chemically bonded to glass beads were chosen. The former support is less suitable for immobilization of oxydases due to strong retention of hydrogenperoxide on the ion exchanger.

The relatively little flow dependence of these systems renders them suitable for low-cost manual sample injection monitors as well as in a flow injection analyses (FIA) mode with low-cost pumping systems. The system was operated with 80% acetonitrile water solutions. A detection limit of 8 × 10^?7M of glucose was achieved in directly injected samples.

Enzymes more sensitive to organic solvents can be operated with pure water and adjustment for optimal chemiluminescence condition is achieved with a make-up flow prior to detection. A detection limit of 5 × 10^?8M glucose is achieved under these conditions. The feasability of this approach to other oxidase based monitors and to detection in liquid chromatography is discussed.     

Amperometric Determination of Galactose,Lactose and Dihydroxyacetone Using Galactose Oxidase in a Flow Injection System with Immobilized Enzyme Reactors and On-Line Dialysis

Abstract

A flow injection manifold containing a dialyzer and reactors with immobilized galactose oxidase and peroxidase was used for the determination of galactose in urine, lactose in milk and dihydroxyacetone in a biotechnological reaction medium. The hydrogen peroxide which is formed by the galactose oxidase reaction was detected by amperometric reduction of a mediator. The latter had been produced from hydrogen peroxide in a peroxidase catalyzed reaction. The hydrogen peroxide detection step was studied with several mediators and hexacyanoferrate (II) was selected. An ion exchange HPLC procedure was used to purify the galactose oxidase, in particular from catalase, and the kinetics and the selectivity of a reactor containing the immobilized enzyme was investigated. Columns for removal of certain interferents such as ascorbic acid were used in the determination of galactose in urine. The response to galactose standards was linear from the detection limit of 2 μM to 60 mM. The throughput was 45 samples per hour and the relative standard deviation 0.4%.