Immobilization of myoglobin on phosphate and phosphonate grafted-zirconia nanoparticles

We investigated the adsorption and catalytic activity of myoglobin (Mb) immobilized on colloidal particles of zirconia covalently grafted with phosphoric (ZrO2-P) and benzenephosphonic acid (ZrO2-BP). The maximum adsorption was reached after 1 h of contact and was greater on a hydrophilic support, ZrO2-P, compared to a hydrophobic support, ZrO2-BP. The equilibrium isotherms fitted the Langmuir equation, suggesting the presence of a monolayer of protein molecules on the surface of the nanoparticles. The nanostructured biocomposites are active in the oxidation of 2-methoxyphenol (guaiacol) by hydrogen peroxide. The oxidation catalyzed by immobilized Mb followed a Michaelis-Menten kinetics, similar to that observed in the oxidation by free Mb. Furthermore, the catalytic efficiency is similar to that of free Mb and higher than that of "large-size" biocatalysts (with sizes larger than 1 mum). In the latter case, the kinetic parameters, k(cat) and K(M), indicate that this is mostly due to an increased affinity of the nano-biocomposite for the substrate. The activity of the nano-biocomposites decreases slightly as the amount of adsorbed protein increases. This is mainly due to the formation of a nonordered monolayer, which reduces the accessibility of the substrate to the active center.     

Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry

A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 degrees C was found to be proportional to the concentration of Mb in the range of 1.0 x 10(-6) to 4.0 x 10(-9)mol L(-1). The detection limit of Mb was found to be 9.93 x 10(-10)mol L(-1). The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.     

层状硅酸盐固定化肌红蛋白在有机溶剂中的催化活性和稳定性

在温和条件(室温及pH=7)下,通过剥离重组实现了肌红蛋白分子在层状硅酸盐magadiite纳米粒子层间的固定化,系统考察了固定化后肌红蛋白在不同疏水度的有机溶剂中催化邻苯二胺氧化反应的性能.结果表明,固定化肌红蛋白在中等疏水度的有机溶剂中呈现出比自由肌红蛋白更高的催化活性和稳定性.结合固定化肌红蛋白在有机溶剂中的动力学研究结果,初步探讨了其具有高催化活性的原因.     

Adsorption of myoglobin onto porous zirconium phosphate and zirconium benzenephosphonate obtained with template synthesis

Porous zirconium phosphate (P-ZrP) and zirconium benzenephosphonate (P-ZrBP) were prepared in the presence of an anionic surfactant acting as a template. Poorly crystalline materials with a P/Zr molar ratio equal to 2 and having a relatively high surface area and micro/mesoporosity have been obtained. The interaction of myoglobin with the two types of surfaces, the hydrophobic P-ZrBP and the hydrophilic P-ZrP, was investigated, and the adsorption isotherms were determined at different pH and temperature values. A model was proposed for the mechanism of the interaction of the protein with the surface based on the shape of the adsorption isotherm and the physical-chemical properties of myoglobin. The pH has been found to be an important parameter for determining the maximum adsorption capacity of P-ZrBP and P-ZrP for myoglobin molecules because of the changes that occur in the type and net charge of the protein surface as the pH of the medium changes. Protein binding affinity and capacity increase when the temperature is increased. This phenomenon occurs because myoglobin varies its conformation at high temperature with an increase in the exposed hydrophobic region. This process causes a stronger hydrophobic interaction between the protein and the adsorbent and reduces the repulsion between the adsorbed molecules. Studies on the activities of the obtained biocomposites are in progress.     

Direct electron-transfer of myoglobin within a new zwitterionic gemini surfactant film and its analytical application for H2O2 detection

The direct electron-transfer of myoglobin in a new zwitterionic gemini surfactant film with glassy carbon electrode surface has been investigated. A pair of well-defined and quasi-reversible voltammetric peaks was observed at −0.34 and −0.30 V due to the direct electron-transfer of the redox couple of Mb (Fe^III/Fe^II). The voltammetric responses of myoglobin–surfactant film under different pH and scan rate conditions were obtained. The presence of hydrogen peroxide changed the typical electrochemical behaviors in terms of bioelectrocatalysis of myoglobin to hydrogen peroxide, and a higher sensitive electroanalytical method for the determination of hydrogen peroxide has been developed.     

Direct electrochemistry and electrochemical catalysis of myoglobin-TiO2 coated multiwalled carbon nanotubes modified electrode

TiO(2) nanoparticles were homogeneously coated on multiwalled carbon nanotubes (MWCNTs) by hydrothermal deposition, and this nanocomposite might be a promising material for myoglobin (Mb) immobilization in view of its high biocompatibility and large surface. The glassy carbon (GC) electrode modified with Mb-TiO(2)/MWCNTs films exhibited a pair of well-defined, stable and nearly reversible cycle voltammetric peaks. The formal potential of Mb in TiO(2)/MWCNTs film was linearly varied in the range of pH 3-10 with a slope of 48.65 mV/pH, indicating that the electron transfer was accompanied by single proton transportation. The electron transfer between Mb and electrode surface, k(s) of 3.08 s(-1), was greatly facilitated in the TiO(2)/MWCNTs film. The electrocatalytic reductions of hydrogen peroxide were also studied, and the apparent Michaelis-Menten constant is calculated to be 83.10 microM, which shows a large catalytic activity of Mb in the TiO(2)/MWCNTs film to H(2)O(2). The modified GC electrode shows good analytical performance for amperometric determination of hydrogen peroxide. The resultant Mb-TiO(2)/MWCNTs modified glassy carbon electrode exhibited fast amperometric response to hydrogen peroxide reduction, long term life and excellent stability. Finally the activity of the sensor for nitric oxide reduction was also investigated.     

QUANTUM chemical estimation of the binding strength of As,Cd, Pb,Sb, Se,Te atoms by the ZrC(100) surface

The publication considers the modifying effect of refractory zirconium carbide in determining elements by hydride generation atomic absorption spectrometry. It is shown that the nature of binding of atoms of the analytes consists in the ability of the ZrC surface to strong adsorption. For As, Cd, Pb, Sb, Se, Te elements the adsorption energies of atoms on the ZrC(100) surface are calculated by the density functional theory. It is found that the necessary condition for adsorption is the preliminary cleaning of the ZrC surface from chemisorbed hydrogen. Correlation is demonstrated between the pyrolysis temperature that is maximum achievable for the analyte in the specimen with the adsorption energy of its atomic form on the ZrC(100) surface.     

Assembly of electroactive layer-by-layer films of myoglobin and ionomer poly(ester sulfonic acid)

Layer-by-layer films were assembled on solid substrates by alternate adsorption of negatively charged ionomer poly(ester sulfonic acid) or Eastman AQ55 from its aqueous dispersion and positively charged myoglobin (Mb) from its solution at pH 4.5. The film assembly process was monitored by cyclic voltammetry (CV), UV-vis spectroscopy, and quartz crystal microbalance (QCM). [AQ/Mb](n) films grown on pyrolytic graphite (PG) electrodes showed a pair of well-defined and nearly reversible CV peaks at about -0.20 V vs Ag/AgCl in pH 5.5 buffers, characteristic of the Mb heme Fe(III)/Fe(II) redox couple. Although the amount of Mb adsorbed in each bilayer was essentially the same, the fraction of electroactive Mb decreased dramatically with an increase of bilayer number (n). Soret absorption bands of [AQ/Mb](n) films on glass slides suggest that Mb in the films retains its native state in the medium pH range. Trichloroacetic acid, oxygen, and hydrogen peroxide were electrochemically catalyzed by [AQ/Mb](6) films with significant lowering of reduction overpotential.     

Activity of copper–cerium–zirconium catalysts in oxidation of hydrogen

We have studied the catalytic properties in oxidation of hydrogen for copper–cerium oxide systems deposited on supports obtained by calcination of yttrium-stabilized zirconium dioxide at 300–1000 °C. We have shown that the catalytic activity of the samples obtained depends on the specific surface area of the original supports and the amount of reduced copper within the composition of the catalyst. In samples whose support has high specific surface area, the content of reduced metallic copper is greater and the catalytic activity is higher.     

Structure, stability, and activity of myoglobin adsorbed onto phosphate-grafted zirconia nanoparticles

The adsorption of myoglobin (Mb) onto phosphate grafted-zirconia (ZrO2-P) nanoparticles was studied in terms of conformational studies and thermal stability, determined by circular dichroism (CD), differential scanning calorimetry (DSC), and atomic force microscopy (AFM). The changes in protein structure have been correlated with the catalytic activity of free and adsorbed Mb. CD and DSC studies indicate marked rearrangements in Mb structure upon adsorption onto phosphate-grafted zirconia nanoparticles. These structural rearrangements of Mb could be responsible for the loss of catalytic activity observed for the adsorbed Mb. In particular, the conformational changes due to the adsorption process induced a reduction of kcat and KM. AFM measurements indicate that the interaction with the grafted-zirconia nanoparticles also affects the morphology of the bound protein, inducing the nucleation of prefibrillar-like aggregates.     

Effect of ZrO<Subscript>2</Subscript> morphology in copper–cerium–zirconium oxide systems on their catalytic properties in the reaction of co oxidation in hydrogen-rich mixtures

We studied the catalytic properties of copper–cerium oxide systems, deposited on supports obtained by calcination of yttrium-stabilized zirconium dioxide at 300-1000 °C, in the reaction of selective oxidation of CO in a stream of hydrogen. We have shown that the catalytic activity of the samples obtained correlates with the activity of the original supports in the reaction of CO oxidation: the highest CO conversion is observed on catalysts with the highest and the lowest specific surface area. Translated from Teoreticheskaya i éksperimental’naya Khimiya, Vol. 45, No. 2, pp. 115-120, March-April, 2009.     

魔芋多糖固定化肌红蛋白的直接电化学与电催化

用魔芋多糖(KGM)和N,N-二甲基甲酰胺(DMF)的加合物,将肌红蛋白(Mb)固定在玻碳电极(GCE)上,制备了稳定的Mb-KGM-DMF/GCE修饰电极,并研究了Mb在修饰电极上的直接电化学行为和电催化性能。该电极在pH=7.0的磷酸盐缓冲溶液(PBS)中,-0.38 V(E0′)处有一对氧化还原峰,峰电位差ΔEp=70 mV,该峰正是Mb中血红素辅基FeⅢ/FeⅡ电对的氧化还原特征峰。在0.2~9.0 V/s扫速的范围内,氧化还原峰峰电流大小和扫描速率成正比,呈现出表面控制行为。在pH为5.0~12.0的范围内,式电位和pH值呈线性关系,表明电子传递过程伴随着质子转移。同时,Mb-KGM-DMF/GCE修饰电极表现出良好的电催化性能,对氧、H2O2有显著的催化作用。在4.70~75.0μmol/L的范围内,其催化峰电流大小与H2O2的浓度有良好的线性关系,其线性回归方程i=0.127 0.093C,r=0.9989,表观米氏常数为80.8μmol/L。     

Nitrite increases the enantioselectivity of sulfoxidation catalyzed by myoglobin derivatives in the presence of hydrogen peroxide

The effect of nitrite in the sulfoxidation of organic sulfides catalyzed by myoglobin (Mb) in the presence of hydrogen peroxide has been investigated. A general improvement in enantioselectivity was found for the reaction catalyzed by horse heart metMb and a series of sperm whale metMb derivatives including the wild type protein, the active site mutants T67K Mb, T67R Mb, T67R/S92D Mb, and the T67K Mb derivative reconstituted with the modified prosthetic group protohemin-l- histidine methyl ester.     

Synthesis of submicrometer zirconium carbide formed from inorganic–organic hybrid precursor pyrolysis

Zirconium carbide (ZrC) was synthesized from inorganic–organic hybrid precursor’s pyrolysis by solution-based processing. Zirconium-containing complexes, which were obtained by chelation of oxide bidentate ligands to zirconium, were used to combine with phenolic resin to form precursors for ZrC. The precursors using specific ligands including acetylacetone, ethyleneglycol, and salicylic acid transformed into pure ZrC at a relatively low temperature (1,550 °C) in addition to that using lactic acid. As a comparison, synthesis of ZrC only using zirconium oxychloride octahydrate (ZrOCl₂·8H₂O) and phenolic resin was also conducted. The synthesized powders had a small average crystallite size (~300 nm), and a low oxygen content (~2.5 at.%). The conversions from as-synthesized preceramic precursors to ceramics were studied by means of FTIR, SEM, EDS, XRD, and XPS. The oxidation behavior of the synthesized ZrC in air was studied by DSC-TG analysis.     

Third generation biosensor based on myoglobin-TiO2/MWCNTs modified glassy carbon electrode

TiO2 nanoparticles were homogeneously coated on multi-walled carbon nanotubes by hydrothermal deposition, this nanocomposite may be a promising material for myoglobin immobilization in view of its high biocompatibility and large surface. The glassy carbon electrode modified with Mb-TiO2/MWCNTs films exhibited a pair of weU defined, stable and nearly reversible cycle voltammetric peaks. The electron transfer between Mb and electrode surface, Ks of 3.08 s^-1, was greatly facilitated in the TiO2/ MWCNTs film. The electrocatalytic reductions of hydrogen peroxide were studied, the apparent Michaelis-Menten constant is calculated to be 83.10 μmol/L, which shows a large catalytic activity of Mb in the TiO2/MWCNTs film to H2O2.     

Direct electrochemistry and electrocatalysis of myoglobin based on silica-coated gold nanorods/room temperature ionic liquid/silica sol-gel composite film

A novel biosensor based on the silica-coated gold nanorods (GNRs@SiO₂) and hydrophilic room temperature ionic liquid (RTIL) 1-butyl-3-methylimidazolium tetrafluroborate ([bmim][BF₄]) was fabricated for the determination of hydrogen peroxide (H₂O₂) and nitrite. GNRs@SiO₂ can not only act as a binder to hinder [bmim][BF₄] (RTIL) leaking from the electrode surface, but also provide a favorable microenvironment for direct electrochemistry of myoglobin (Mb). A pair of well-defined and quasi-reversible redox peaks of Mb was obtained at the GNRs@SiO₂-Mb/RTIL-sol-gel composite film modified GCE (GNRs@SiO₂-Mb/RTIL-sol-gel/GCE) through direct electron transfer between Mb and the underlying electrode. This biosensor showed an excellent electrocatalytic activity towards hydrogen peroxide and nitrite. The linear range for the determination of H₂O₂ was from 0.2 to 180 μM with a detection limit of 0.12 μM based on the signal-to-noise ratio of 3. In addition, the biosensor also exhibited high selectivity, good reproducibility, and long-term stability. Therefore, this kind of composite film can provide an ideal matrix for protein immobilization and biosensor fabrication.     

A kinetic-spectrophotometric method for the determination of glucose in solutions

A kinetic-spectrophotometric method is proposed to determine glucose in solutions. Measurements were performed at 400 nm; the negative peak was obtained by subtracting the absorption spectra of myoglobin (Mb) before and after oxidation. In this method, glucose is added to a mixture of Mb and glucose oxidase. Glucose is oxidized by glucose oxidase and oxygen to gluconate and hydrogen peroxide is generated. The liberated hydrogen peroxide oxidizes the Mb heme (Fe²⁺) into Fe³⁺. The higher the glucose concentration added, the more the H₂O₂ generation, and the more the Mb oxidation (Fe²⁺ to Fe³⁺) and, as a result, the higher the absorbance at 400 nm (negative peak, lower absorbance value). The increments of added glucose are monitored by measuring the absorbance decay versus time (0–250 s) at 400 nm. Each glucose concentration has an accompanying unique absorbance value at 250 s. The higher the glucose concentrations, the lower the absorbance at 250 s (measured at 400 nm). The calibration curve for glucose was linear from 0.1 to 3.0 mM; the detection limit was found to be 0.025 mM. There was no interference from major substances present; the only interference was from species that react with H₂O₂ (ascorbic acid, uric acid, and urea) or that react with glucose (Cu²⁺ and Fe³⁺). Standard deviation in the determination was ±0.01 mM for a 1.3 mM glucose solution (n = 10). The text was submitted by the author in English.     

Copper catalysts based on fiberglass supports for hydrocarbon oxidation reactions with the participation of hydrogen peroxide

Copper-containing catalysts were prepared by the adsorption of the ammonia complexes of Cu(II) on the surface of a silicate fiberglass material followed by the thermal and oxidative treatment of the samples. The states of copper after the adsorption of ammonia complexes and in the prepared samples were characterized using electronic diffuse reflectance spectroscopy. The catalytic activity of the samples in hydrogen peroxide decomposition and cyclohexane oxidation reactions was studied. It was found that molecular oxygen can be involved in the radical process of hydrogen peroxide oxidation. Based on spectroscopic data, it was hypothesized that partially reduced Cu(I)–Cu(O) compounds are active species in the catalysts of this type.     

Ultrathin layered myoglobin-polyion films functional and stable at acidic pH values

Cross-linking of myoglobin (Mb) promoted by 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide within films of polystyrene sulfonate after layer-by-layer self-assembly provided remarkable stabilization. Cross-linking greatly improved adhesion of the films to fused silica slides and allowed extensive optical studies over a wide pH range. Circular dichroism and visible absorbance spectra showed that Mb retained its native conformation when films were placed in solutions of pH as low as 2 and up to pH 11. Linear dichroism revealed an average orientation of the Mb iron heme cofactors of 58 degrees to the film normal. High concentrations of urea did denature the protein in the films, however. At pH 1, Mb in solution is fully unfolded but retained considerable alpha-helical content in the cross-linked films. Both the polyion film environment and cross-linking seem to play roles in stabilizing protein secondary structure and function at low pH. Cross-linked myoglobin-polyion films on pyrolytic graphite electrodes were used in strongly acidic solutions for the electrochemical catalytic reduction of trichloracetic acid, hydrogen peroxide, and oxygen. The pH-dependent catalytic reduction of trichloracetic acid was faster in 0.1 M HCl than in the medium pH range.     

Direct electrochemistry and electrocatalysis of heme-protein based on N,N-dimethylformamide film electrode

The heme-protein including myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP) were immobilized on normal graphite electrode by using N,N-dimethylformamide (DMF). The proteins undergo direct electron-transfer reactions. The current is linearly dependent on the scan rate, indicating that the direct electrochemistry of heme-protein in that case is a surface-controlled electrode process. The E°s are linearly dependent on solution pH (redox-Bohr effect), indicating that the electron transfer was proton-coupled. Ultraviolet-visible (UV-vis) and reflection-absorption infrared (RAIR) spectra suggest that the conformation of proteins in the presence of DMF are little different from that proteins alone the conformation changes reversibly in the range of pH 3.0-10.0. The catalytic activity of proteins were examined by hydrogen peroxide and nitrite.