Comparative study of the inactivation of horseradish peroxidase under the effect of H2O2 and ionizing radiation

The inactivation of native and recombinant horseradish peroxidase in the presence of hydrogen peroxide and under ionizing radiation was studied. The types of peroxidase activity differ in sensitivity towards the inactivating effect of H₂O₂: the activity in relation to the iodide ion is more stable than the activity in relation to ammonium 2,2-azinobis(3ethylbenzothiazoline-6-sulfonate) (ABTS) ando-phenylenediamine. Similar inactivation was observed in the course of the radiolysis of peroxidase. It was assumed that the initial period of peroxidase inactivation in the presence of hydrogen peroxide has a radical nature and is related to the generation of Superoxide radicals, which modify the protein moiety, resulting in the destruction of heme. The R-670 compound was not formed under the conditions studied. However, the E EI transition occurred, depending on the radiation dose and the enzyme concentration.Translated fromIzyestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 176–179, January, 1995.     

Comparative studies of stability of native and recombinant horseradish peroxidase inactivated by radiation and other factors

Comparative studies of the inactivation of native and recombinant horseradish peroxidase in the course of an enzymatic reaction, at elevated temperatures and in a wide range of radiation doses, have been performed. The protective effect of the carbohydrate component of the native peroxidase providing for stabilization of the enzyme against various inactivating factors was demonstrated. It was proposed that radioactive inactivation is related to dysfunction in heme interaction with the protein component and to an increase in the conformational mobility around the active site of the enzyme.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2230–2233, December, 1994.     

The role of tryptophan residues in radiation sensitivity of enzymes as exemplified in horseradish peroxidase

The stability of recombinant wild-type horseradish peroxidase and its tryptophan-less mutant Trp117Phe toward γ-radiation was studied. The absence of tryptophan in the enzyme molecule results in a certain stabilization, which is manifested as the absence of the initial drop in activity and appearance of a lag period for doses of up to 45 Gy. Contrary to the wild-type enzyme, the dose response of the mutant is almost independent of the nature of the substrate used to measure the catalytic activity; this indirectly indicates that Trp117 participates in the oxidation of substrates. Pretreatment of the wild-type recombinant enzyme with hydrogen peroxide destabilizes the enzyme towards irradiation, while the same procedure for the mutant enzyme has virtually no effect on the dose response curve. This suggests the modification of Trp117 in the oxidation of the native enzyme with H₂O₂ in the absence of electron-donor substrates, which is the modification of Trp171 in the recombinant lignin peroxidase. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2355–2358, December, 1999.     

Catalytic properties of Phe41→His mutant of horseradish peroxidase expressed inE. coli

The recombinant horseradish peroxidase and its single-point F41H mutant have been reactivated fromE. coli inclusion bodies. The influence of the mutation on the heme entrapment, stability and activity of the enzyme was demonstrated. The catalytic rate constants for H₂O₂ cleavage and ammonium 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) oxidation decrease by two and one orders of magnitude, respectively. Unlike the wild-type recombinant horseradish peroxidase, the elimination of the ABTS oxidation product is not a rate-determining step for the mutant. The F41H replacement results in significant changes of kinetics of iodide ion oxidation. The reaction rate is linear to the concentrations of iodide, H₂O₂, and the enzyme. The results suggest the direct interaction of iodide with the porphyrin ring of the heme. The decrease in ABTS oxidation activity accompanied by retention of activity in iodide oxidation in the course of low-dosage radiolysis of the F41H mutant is additional evidence of the direct electron transfer from iodide to the heme, in contrast to ABTS oxidation, in which the electron transfer chain in the protein molecule is involved.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2034–2038, November, 1994.     

Comparative study of horseradish mutant forms by radioenzymology

Radioenzymology was used to study the recombinant and mutant forms of horseradish peroxidase, namely, F221W, Q176E, Q176A, S35K, E64P, E64S, S35A, and S35KQ176E. Both removal of the Trp residue and introduction of an additional one result in a simpler dose response; the insertion of polar residues stabilizes the enzyme molecule through realization of a more closed conformation. The greatest oscillation changes were found for the replacement by Ala. It was assumed that the binding site of guaiacol as a substrate is located near the residue 64, which is structurally related to the residue 176 and the heme. A scheme of formation of the intermediate through rotation of the Trp aromatic ring was proposed.     

Potential control of horseradish peroxidase immobilization on gold electrode

Based on the self-assembled monolayer (SAM) technique, a number of methods for immobilizing protein onto electrodes have been recently reported, such as entrapment method in which the protein was wrapped with regenerated silk fibroin[1], self-assem- bled monolayer[2] and silica sol-gel[3], layer-by-layer self-assembly method in which the protein was ad-sorbed to opposite charged macromolecules due to electrostatic attraction[4], reversed micelle[5], cross- linked method[6] and surface spin-coa…     

Layer-by-layer immobilization of horseradish peroxidase on a gold electrode modified with colloidal gold nanoparticles

An amperometric biosensor, based on layer-by-layer self-assembly of colloidal gold nanoparticles, cysteine and horseradish peroxidase on Nafion modified electrode surface by electrostatic adsorption, has been used for the determination of hydrogen peroxide. The electrochemical behavior of the multilayer film was studied by cyclic voltammetry, linear sweep voltammetry and chronoamperometry. The step layer-by-layer adsorption interface morphology was further characterized by means of electrochemical impedance spectroscopy and cyclic voltammetry. The performance and factors influencing the resulted biosensor were studied in detail. The sensor displayed an excellent electrocatalytic response to the reduction of H₂O₂ without the aid of an electron transfer mediator. Linear response to H₂O₂ was obtained for the concentration range from 1.6 μM to 2.4 mM under optimized conditions. The detection limit of the biosensor was 0.5 μM (S/N = 3), and the sensor achieved 95% of the steady-state current within 10 s. The sensor exhibited high sensitivity, selectivity and stability. Correspondence: Yan Liu, College of Chemistry, Chongqing Normal University, Chongqing 400047, P.R. China     

Oxidative polymerization of phenylenediamines catalyzed by horseradish peroxidase

Ortho-, meta-, and para-phenylenediamines were polymerized using hydrogen peroxide as an oxidant and horseradish peroxidase as a catalyst in mixed solvents of 1,4-dioxane and water. The yield of the polymers was strongly dependent on solvent composition, and maximum yields were obtained at 15–30% 1,4-dioxane. The analysis of circular dichroic spectra of the enzyme suggested that enzyme structure was significantly modified at high 1,4-dioxane contents, which may be responsible for the decrease of catalytic activity of the enzyme. On the basis of IR and electronic spectra of the polymers, it was considered that o- and p-phenylenediamine polymers retain disubstituted benzene nuclei, which suggests that the polymerization proceeded mainly via N—N coupling. © 1998 John Wiley & Sons, Inc. J. Polym. Sci. A Polym. Chem. 36: 2593–2600, 1998     

氧化石墨烯在辣根过氧化物酶传感器中的应用研究

本文研究氧化石墨烯的合成方法及其在生物传感器中的应用.通过Hummer法氧化天然石墨粉制得氧化石墨,在蒸馏水中利用超声分散将氧化石墨剥片,从而合成了氧化石墨烯(GO).通过透射电镜图表征了氧化石墨烯的形貌并通过红外光谱证实氧化石墨烯的形成.将所合成的氧化石墨烯与三角形貌的金纳米颗粒(prism AuNPs)、辣根过氧化...     

Effects of sodium phosphate buffer on horseradish peroxidase thermal stability

Thermal stability of horseradish peroxidase (HRP) was studied by differential scanning calorimetry, tryptophan fluorescence, the heme absorption and enzymatic activity analysis while the concentrations of sodium phosphate buffer ranged from 2.5 to 50 mM at pH 7.0. The results showed that the denaturation temperature (T _m) values decreased and the intrinsic tryptophan fluorescence intensity of denatured HRP increased as sodium phosphate buffer concentration increased. Furthermore, the heme absorbance at 403 nm and enzymatic activity of HRP decreased with the increasing buffer concentrations. According to data obtained in this experiment, it can be concluded that sodium phosphate accelerated the denaturation process of HRP and reduced the thermal stability of HRP.     

Electrochemical behavior of horseradish peroxidase on WS2 nanosheet‐modified electrode and electrocatalytic investigation

In this paper, a WS₂ nanosheet was modified on the surface of a carbon ionic liquid electrode (CILE), and horseradish peroxidase (HRP) was further fixed on the electrode with a Nafion film. Direct electrochemistry and bioelectrocatalysis of HRP incorporated on the modified electrode were investigated in detail. On Nafion/HRP/WS₂/CILE, a pair of well‐defined quasi‐reversible redox peaks appeared on the cyclic voltammogram, indicating that the presence of the WS₂ nanosheet on the electrode surface could provide a specific interface with large surface area for HRP and its direct electron transfer rate was greatly enhanced. The formal potential (E⁰) obtained was –0.179 V, which was the typical feature of heme Fe(III)/Fe(II) in HRP. The electron transfer coefficient (α) and the heterogeneous electron transfer rate constant (k_s) of HRP were calculated as 0.44 and 1.01 s^–1, respectively. This HRP‐modified electrode showed excellent electrocatalytic activity for the reduction of trichloroacetic acid and NaNO₂ with a wide linear range and low detection limit. Real samples were detected by this proposed method, indicating the successful fabrication of a new third‐generation electrochemical enzyme sensor utilizing the WS₂ nanosheet.     

Structure and properties of the graft copolymer of starch and p‐hydroxybenzoic acid using horseradish peroxidase

A modified starch tannage was synthesized by free radical graft copolymerization of degraded starch with p‐hydroxybenzoic acid (pHA) using horseradish peroxidase/H₂O₂ as the initiator. In this study, the effects of the degree of degradation of the starch, dosage of pHA, polymerization temperature, system pH, and horseradish peroxidase content on the tanning properties of the graft copolymer were investigated. The shrinkage temperature of leather tanned by the graft copolymer was 78 °C. The thickness increment ratio of the retanned leather was 21.6%, and meanwhile the retanned leather showed better softness. The results indicate that the graft copolymer has excellent tanning and retanning properties. The structure of the graft copolymer was analyzed by Fourier Transform Infrared Spectroscopy (FTIR), ¹Hydrogen‐Nuclear Magnetic Resonance (¹H‐NMR), and ¹³Carbon‐Nuclear Magnetic Resonance (¹³ C‐NMR) and Gel Permeation Chromatography (GPC). Compared with conventional methods, we show that a “green” leather tannage could be achieved using a radical graft copolymerization of starch and phenols. Copyright © 2011 John Wiley & Sons, Ltd.     

生物酶HRP催化H~2O~2氧化间苯二胺反应的研究

应用电化学分析,高效液相色谱(HPLC),紫外-可见光谱(UV-vis),红外光谱(IR)和核磁共振(NMR)等技术对辣根过氧化物酶(HRP)催化H~2O~2氧化间苯二胺(MPD)的反应进行了研究。伏安法和高效液相色谱实验说明,在所选择的酶催化反应条件下,酶催化反应生成一种产物。用化学方法制得了HRP酶催化H~2O~2氧化MPD的产物纯品。经UV-vis,IR和^1HNMR谱鉴定,产物为2,7-二氨基吩嗪。写出了酶催化反应过程,同时对酶催化反应产物的电极还原过程也进行了研究。     

基于氧化还原反应界面可视化定量检测辣根过氧化物酶

该文建立了一种可视化的、基于氧化还原反应界面移动距离定量检测辣根过氧化物酶(HRP)的方法。比较了隐色结晶紫显色体系和3,3′,5,5′-四甲基联苯胺显色体系对HRP的显色效率,并构建了基于3,3′,5,5′-四甲基联苯胺显色体系的氧化还原反应电泳滴定模型。同时,文中还设计了适用于该模型的小型化、便携式滴定检测芯片,并对滴定通道凝胶中组分进行了优化。结果表明,界面移动距离与HRP浓度存在对数线性关系,检测灵敏度可达0.002 mg/L,且可在10 min内完成HRP的裸眼检测。该方法不需要配备信号读取装置,用户只需要读取有色界面移动的距离即可实现对待测物的可视化定量检测,对于即时检测具有潜在的应用价值。     

Enzymatic oxidative polymerization of para‐imine functionalized phenol catalyzed by horseradish peroxidase

Enzymatic oxidative polymerization of a new para‐imine functionalized phenol derivative, 4‐(4‐hydroxybenzylideneamino)benzoic acid (HBBA), using horseradish peroxidase enzyme and hydrogen peroxide oxidizer has been investigated in an equivolume mixture of an organic solvent (acetone, methanol, ethanol, dimethylformamide, 1,4‐dioxane, and tetrahydrofuran) and phosphate buffer (pH = 5.0, 6.0, 6.8, 7.0, 7.2, 8.0, and 9.0) at different temperatures under air for 24 h. The resulting oligomer, oligo(4‐(4‐hydroxybenzylideneamino)benzoic acid) [oligo(HBBA)], was characterized using ultraviolet–visible, Fourier transform infrared (FT‐IR), ¹H nuclear magnetic resonance (NMR), cyclic voltammetry, size exclusion chromatography, differential scanning calorimetry, and thermogravimetric analyses. Polymerization involved carbon dioxide and hydrogen elimination from the monomer, and terminal units of the oligomer structure consisted of phenolic hydroxyl (–OH) groups at the ends. The polymer is mainly composed of a mixture of phenylene and oxyphenylene units according to ¹H NMR and FT‐IR analyses. Effects of solvent system, temperature and buffer pH on the polymerization have been investigated in respect to the yield and molecular weight (M_n) of the product. The best condition in terms of the highest molecular weight (M_n = 3000 g/mol, DP ~ 15) was achieved in an equivolume mixture of 1,4‐dioxane/pH 5.0 phosphate buffer condition at 35°C. Electrochemical characterization of oligo(HBBA) was investigated at different scan rates. The resulting oligomer has also shown relatively high thermal stability according to thermogravimetric analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of tetrabutylammonium bromide on enzymatic polymerization of phenol catalyzed by horseradish peroxidase

In this article tetrabutylammonium bromide （TBAB） was first added in buffer to compose a convenient and environmentally friendly system, and enzymatic polymerization of phenol catalyzed by horseradish peroxidase （HRP） could proceed efficiently in this system. When TBAB was added, the most conversion of phenol could reach 99.1%. The phenol polymer was considered to consist of a mixture of phenylene （Ph） and oxyphenylene （Ox） units by IR analysis, and the ratio of phenylene to oxyphenylene units （Ph/Ox） was measured by titration. Moreover, the effects of the dosage of horseradish peroxidase （HRP） and pH value on the conversion of phenol were investigated. The reaction performed very effectively in this novel system when the addition of HRP was only 0.2 mg. In all cases, the weight-average molecular weight calculated by GPC-SLS was in a range from 12000 Da to 30000 Da. The phenol polymer prepared in the present research possessed good thermal stability shown by TG analysis.     

Fibre optic fluorometric enzyme sensors for hydrogen peroxide and lactate,based on horseradish peroxidase and lactate oxidase

An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H₂O₂. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum.     

Photoinitiated oxidation of NADH catalyzed by horseradish peroxidase studied by chemically induced dynamic nuclear polarization

The photoinitiated oxidation of β-NADH catalyzed by horseradish peroxidase (Per³⁺) was studied by time-resolved photoinitiated chemically induced dynamic nuclear polarization (CIDNP). The polarization observed on protons at the C(4) atom of the β-NADH molecule is evidence for the reversible one-electron transfer between the radical cation NADH^+· and the ferroperoxidase intermediate (Per²⁺). A new approach based on electron transitions in the (NADH^+· Per²⁺) pair was proposed to describe the formation of CIDNP effects in systems including quartet (Q)—doublet (D) electron transitions. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1090–1094, July, 2006.     

Changes in substrate specificity of native and recombinant horseradish peroxidase under ionizing radiation

The homogeneous recombinant horseradish peroxidase preparation fromE. coli inclusion bodies exhibits higher specific activity towards ammonium 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) than the native one. The differences in substrate specificity can be assigned to the native enzyme inactivation in the course of metabolic reactions in living plant cells, while the recombinant enzyme reconstructedin vitro completely realizes the original catalytic abilities. Application of the method of radiation-induced inactivation demonstrates the existence of different binding sites for the iodide anion. ABTS, phenol, and guaiacol and allows one to assume a common character of the binding sites of phenol ando-phenylenediamine.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2234–2237, December, 1994.     

Wild type and mutant forms of recombinant horseradish peroxidase C expressed inEscherichia coli

Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41→ His and Phel43→ Glu, were compared to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation. Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism. Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring. The replacement of Phe41 with the ionizable His residue destabilizes the enzyme. The Phel43→ Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates and free radicals. The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2′-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaiacol, and o-phenylene diamine. The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring. The results also suggest that the ABTS binding site is less accessible than that for o-phenylene diamine.