Direct electrochemistry and bioelectrocatalysis of horseradish peroxidase immobilized on active carbon

For the first time horseradish peroxidase (HRP) immobilized on the surface of active carbon powder modified at the surface of a glassy carbon electrode has been shown to undergo a direct quasi-reversible electrochemical reaction. Its formal potential, E-o/, is -0.363 V in phosphate buffer solution (pH 6.8) at a scan rate of 100 mV/s and is almost independent of the scan rate in the range of 50-700 mV/s. The dependence of E-o/ on the pH of the buffer solution indicated that the conversion of HRP-Fe(III)/HRP-Fe(II) is a one-electron-transfer reaction process coupled with one-proton-transfer. The experimental results also demonstrated that the immobilized HRP retained its bioelectrocatalytic activity to the reduction of H2O2. Furthermore, the HRP adsorbed oil the surface of the active carbon powder can be stored at 4 degreesC for several months without any loss of the enzyme activity. The method presented for immobilizing HRP can be easily extended to immobilize and obtain the direct electrochemistry of other enzymes.

Direct electrochemical reaction of horseradish peroxidase immobilized on the surface of active carbon powders

It is reported for the first time that horseradish peroxidase (HRP) immobilized on the active carbon can undergo a direct quasi-reversible electrochemical reaction. In addition, the immobilized HRP showed the stable bioelectrocatalytic activity for the reduction of H2O2.

A novel hydrogen peroxide sensor based on horseradish peroxidase immobilized in DNA films on a gold electrode

A novel third-generation hydrogen peroxide (H2O2) biosensor was developed by immobilizing horseradish peroxidase (HRP) on a biocompatible gold electrode modified with a well-ordered, self-assembled DNA film. Cysteamine was first self-assembled on a gold electrode to provide an interface for the assembly of DNA molecules. Then DNA was chemisorbed onto the self-assembled monolayers (SAMs) of cysteamine to form a network by controlling DNA concentration. The DNA-network film obtained provided a biocompatible microenvironment for enzyme molecules, greatly amplified the coverage of HRP molecules on the electrode surface, and most importantly could act as a charge carrier which facilitated the electron transfer between HRP and the electrode. Finally, HRP was adsorbed on the DNA-network film. The process of the biosensor construction was followed by atomic force microscopy (AFM). Voltammetric and time-based amperometric techniques were employed to characterize the properties of the biosensor derived. The enzyme electrode achieved 95% of the steady-state current within 2 s and had a 0.5 mu mol l(-1) detection limit of H2O2. Furthermore, the biosensor showed high sensitivity, good reproducibility, and excellent long-term stability.

Synthesis and catalytic kinetics of tellurium-modified hyaluronic acid with glutathione peroxidase activity

A novel mimic TeHA was synthesized by modifying hyaluronic acid (HA) with tellurium, whose function is similar to that of glutathione peroxidase (GPX). The structure of TeHA was characterized by means of infrared spectroscopy and nuclear magnetic resonance spectroscopy, showing that the target Te is located at -CH2OH of the N-acetyl-D-glucosamine of HA. The activity of TeHA is 163.6 U/mu mol according to Wilson's method. In contrast to other mimics, TeHA displays a high activity. Moreover, TeHA can use many hydroperoxides as substrates, such as H2O2, cumenyl hydroperoxide, and tert-butyl hydroperoxide, and cumenyl hydroperoxide is the optimal substrate. A ping-pong mechanism was deduced for the reduction reactions catalyzed by TeHA according to the steady-state kinetic studies.

Synthesis and kinetics of a novel mimic with glutathione peroxidase activity - Tellurium-containing hyaluronic acid (TeHA)

A novel mimic was synthesized by modifying hyaluronic acid (HA) with tellurium, whose function is similar to that of glutathione peroxidase (GPX). The structure of TeHA was characterized by means of IR and NMR, the target-Te was located at -CH2OH of the N-acetyl-D-glucosamine of HA. The H2O2 reducing activity of TeHA, by glutathione (GSH), was 163.6 U/mu mol according to Wilson's method. In contrast to other mimics, TeHA displayed the highest activity. Moreover, TeHA accepted many hydroperoxides as its substrates, such as H2O2, cumenyl hydroperoxide (CuOOH) and tert-butyl hydroperoxide (t-BuOOH), and CuOOH was the optimal substrate of TeHA. A ping-pong mechanism was observed in the steady-state kinetic studies of the reactions catalyzed by TeHA.

Lipid membrane immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

Stable films of didodecyldimethylammonium bromide (DDAB, a synthetic lipid) and horseradish peroxidase (HRP) were made by casting the mixture of the aqueous vesicle of DDAB and HRP onto the glassy carbon (GC) electrode. The direct electron transfer between electrode and HRP immobilized in lipid film has been demonstrated. The lipid films were used to supply a biological environment resembling biomembrane on the surface of the electrode. A pair of redox peaks attributed to the direct redox reaction of HRP were observed in the phosphate buffer solution (pH 5.5). The cathodic peak current increased dramatically while anodic peak decreased by addition of small amount H2O2. The pH effect on amperometric response to H2O2 was studied. The biosensor also exhibited fast response (5 s), good stability and reproducibility.

Effect of lanthanum ion on peroxidase in plant

The interaction of MP-11 as a model of antioxidatase enzymes with La3+ was investigated. It was found that La3+ can increase in the non-planarity of heme and the content of alpha helix and beta turn conformations of the MP11 molecule. The change in the secondary structure of the MP-11 molecule can increase in the exposure extent of heme to the solution. Therefore, the electrochemical reaction of MP-11 is promoted and the electrocatalytic activity to the reduction of H2O2 is increased. The results are consistent with that for the interaction of peroxidases(POD), one of the antioxidatase enzymes, obtained in the living plant experiments at low concentration of La3+.

Conformation change of horseradish peroxidase in lipid membrane

The electrochemical behavior of horseradish peroxidase (HRP) in the dimyristoyl phosphatidylcholine (DMPC) bilayer on the glassy carbon (GC) electrode was studied by cyclic voltammetry. The direct electron transfer of HRP was observed in the DMPC bilayer. Only a small cathodic peak was observed for HRP on the bare GC electrode. The electron transfer of HRP in the DMPC membrane is facilitated by DMPC membrane. UV-Vis and circular dichroism (CD) spectroscopy were used to study the interaction between HRP and DMPC membrane. On binding to the DMPC membrane the secondary structure of HRP remains unchanged while there is a substantial change in the conformation of the heme active site. Tapping mode atomic force microscopy (AFM) was first applied for the investigation on the structure of HRP adsorbed on supported phospholipid bilayer on the mica and on the bare mica. HRP molecules adsorb and aggregate on the mica without DMPC bilayer. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed in the DMPC bilayer. The adsorption of HRP in the DMPC bilayer changes drastically the domains and defects in the DMPC bilayer due to a strong interaction between HRP and DMPC films.

A method to construct a third-generation horseradish peroxidase biosensor: Self-assembling gold nanoparticles to three-dimensional sol-gel network

A novel method for fabrication of horseradish peroxidase biosensor has been developed by self-assembling gold nanoparticles to a thiol-containing sol-gel network. A cleaned gold electrode was first immersed in a hydrolyzed (3-mercaptopropyl)-trimethoxysilane (MPS) sol-gel solution to assemble three-dimensional silica gel, and then gold nanoparticles were chemisorbed onto the thiol groups of the sol-gel network. Finally, horseradish peroxidase (HRP) was adsorbed onto the surface of the gold nanoparticles. The distribution of gold nanoparticles and HRP was examined by atomic force microscopy (AFM). The immobilized horseradish peroxidase exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide. The performance and factors influencing the performance of the resulting biosensor were studied in detail. The resulting biosensor exhibited fast amperometric response (2.5 s) to H2O2. The detection limit of the biosensor was 2.0 mumol L-1, and the linear range was from 5.0 mumol L-1 to 10.0 mmol L-1. Moreover, the studied biosensor exhibited high sensitivity, good reproducibility, and long-term stability.

A chemiluminescence optical fiber glucose biosensor based on co-immobilizing glucose oxidase and horseradish peroxidase in a sol-gel film

An optical fiber bienzyme sensor based on the luminol chemiluminescent reaction was developed and demonstrated to be sensitive to glucose. Glucose oxidase (GOD) and horseradish peroxidase (HRP) were co-immobilized by microencapsulation in a sol-gel film derived from tetraethyl orthosilicate(TEOS). The calibration plots for glucose were established by the optical fiber glucose sensor fabricated by attaching the bienzyme silica gel onto the glass window of the fiber bundle. The linear range was 0.2-2 mmol/L and the detection limit was approximately 0.12 mmol/L. The relative standard deviation was 5.3% (n = 6). The proposed biosensor was applied to glucose assay in ofloxacin injection successfully.

Studies of soluble human catalytic antibody with glutathione peroxidase activity

By screening the phage-displayed human single chain antibody library, we have got the specific single chain antibody bound to GSH-S-DNP butyl ester as the hapten. The tertiary structure of the protein was analyzed with the aid of computer, and the results showed the CDR3 region located on the surface of the antibody. The soluble antibody was expressed in E. coli. and the active site serine was converted into selenocysteine with the chemical modifying method, which resulted in the catalytic antibody with GPx activity of 80 U/mu mol. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody was studied.

Investigation of thermal unfolding process of cyanic adduct of horseradish peroxidase by Fourier transform infrared and circular dichroism spectrometry

Detailed circular dichroism(CD) and Fourier transform infrared (FTIR) studies have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme C(HRP) inhibited by CN(HRP-CN). The results suggest that HRP-CN is quite different from native HRP with different spin states of Fe of heme and different coordinated states. Cyanide becomes the sixth ligand of Fe(I) of heme and the hydrogen-binding network is destroyed partly at the same time, which cause the drastic decrease of thermal stability of HRP. The FTIR and Soret-CD spectra analysis demonstrate that during the heating process there is an intermediate state(I') which has both partly destroyed secondary and tertiary structures of native HRP, then it is the appearance of protein aggregation state(A) after fully unfolding. The unfolding pathway thus can be shown as follows: I -->I'-->U -->A.

Human catalytic antibodies with glutathione peroxidase activity

In order to generate catalytic antibodies with glutathione peroxidase (GPx) activity, we prepared GSH-S-DNP butyl ester and GSH-S-DNP benzyl ester as the haptens. Two ScFvs that bound specifically to the haptens were selected from the human phage-displayed antibody library. The two ScFv genes were highly homologous, consisting of 786 bps and belonging to the same VH family-DP25. In the premise of maintaining the amino acid sequence, mutated plasmids were constructed by use of the mutated primers in PCR, and they were over-expressed in E. coli. After the active site serine was converted into selenocysteine with the chemical modifying method, we obtained two human catalytic antibodies with GPx activity of 72.2U/mu mol and 28.8U/mu mol, respectively. With the aid of computer mimicking, it can be assumed that the antibodies can form dimers and the mutated selenocysteine residue is located in the binding site. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody with the higher activity was studied. (C) 2001 Elsevier Science BY. All rights reserved.

Acid-stable amperometric soybean peroxidase biosensor based on a self-gelatinizable grafting copolymer of polyvinyl alcohol and 4-vinylpyridine

A peroxidase was extracted from Chinese soybean seed coat, and its thermostability and acid-stability were characterized. This peroxidase was immobilized into a self-gelatinizable grafting copolymer of polyvinyl alcohol with 4-vinylpyridine(PVA-g-PVP) to construct an acid-stable hydrogen peroxide biosensor. The effect of pH was studied for optimum analytical performances by amperometric and spectro-photometric methods, also the K-m(app) and the stability of the soybean peroxidase-based biosensor are discussed. At pH 3.0, the soybean peroxidase maintained its bioactivity and the enzyme electrode had a linear range from 0.01 to 6.2 mM with a detection limit of 1.0 x 10(-7) M. In addition, the main characteristics of different hydrogen peroxide sensors were compared.

Sol-gel thin-film immobilized soybean peroxidase biosensor for the amperometric determination of hydrogen peroxide in acid medium

An acid-stable soybean-peroxidase biosensor was devel oped by immobilizing the enzyme in a sol-gel thin film. Methylene blue was used as a mediator because of its high electron-transfer efficiency. The sol-gel thin film and enzyme membrane were characterized by FT-IR, and the effects of pH, operating potential, and temperature were explored for optimum analytical performance by using the amperometric method. The H2O2 sensor exhibited a fast response (5 s), high sensitivity (27.5 mu A/mM), as well as good thermostability and long-term stability. In addition, the performance of the biosensor was investigated using flow-injection analysis (FIA).