Hydrazines probe of interaction of horseradish peroxidase with cryo-hydrogel

The interaction between horseradish peroxidase (HRP) and the cryo-hydrogel was probed by using hydrazines which show high specificity of the reaction of the edge in the prosthetic heme of horseradish peroxidase. For comparison, the interaction of hydrazine with the horseradish peroxidase adsorbed on graphite electrode was also carried out by using steady-state response of the enzyme electrode and cyclic voltammetry. In order to obtain a proper explanation of the kinetic parameters for the enzymatic reaction, the theoretical expressions of I-max and K-M' in the Michaelis-Menten equation for the experimental system were provided. (C) 1997 Elsevier Science B.V.

The electrochemical study of oxidation-reduction properties of horseradish peroxidase

Oxidation-reduction properties of horseradish peroxidase (HRP) have been investigated by using direct electrochemical methods. Two successive separated distinct one-electron processes of HRP were obtained and the related physiological processes were described. The monolayer coverage of HRP at the electrode surface is about 50 pmol/cm(2). UV-Vis spectrophotometry and stable amperometry prove that the enzyme electrode possesses catalytic activity for H2O2 in the absence of a mediator and it might offer an opportunity to build the third generation of biosensors for analytes, such as H2O2, glucose and cholesterol etc. (C) 1997 Elsevier Science S.A.

Viologen-thiol self-assembled monolayers for immobilized horseradish peroxidase at gold electrode surface

A stable, well-behaved self-assembled monolayer (SAM) of viologen-functionalized thiol was used to immobilize and electrically connect horseradish peroxidase (HRP) at gold electrode. Viologen groups in SAMs facilitated the electron transfer from the electrode to the protein active site so that HRP exhibited a quasi-reversible redox behavior. HRP adsorbed in the SAMs is very stable, and close to a monolayer with the surface coverage of 6.5 x 10(-11) mol/cm(2). The normal potential of HRP is -580 mV vs Ag/AgCl corresponding to ferri/ferro active center and the standard electron transfer rate constant is 3.41 s(-1) in 0.1 M phosphate buffer solution (pH 7.1). This approach shows a great promise for designing enzyme electrodes with other redox proteins and practical use in tailoring a variety of amperometric biosensor devices. Copyright (C) 1997 Elsevier Science Ltd.

In situ electrochemical scanning tunnelling microscopy investigation of structure for horseradish peroxidase and its electrocatalytic property

Immobilization of protein molecules is a fundamental problem for scanning tunnelling microscopy (STM) measurements with high resolution. In this paper, an electrochemical method has been proved to be an effective way to fix native horseradish peroxidase (HRP) as well as inactivated HRP from electrolyte onto a highly oriented pyrolytic graphite (HOPG) surface. This preparation is suitable for both ex situ and in situ electrochemical STM (ECSTM) measurements. In situ STM has been successfully employed to observe totally different structures of HRP in three typical cases: (1) in situ ECSTM reveals an oval-shaped pattern for a single molecule in neutral buffer solution, which is in good agreement with the dimension determined as 6.2 x 4.3 x 1.2. nm(3) by ex situ STM for native HRP; (2) in situ ECSTM shows that the adsorbed HRP molecules on HOPG in a denatured environment exhibit swelling globes at the beginning and then change into a V-shaped pattern after 30 min; (3) in situ ECSTM reveals a black hole in every ellipsoidal sphere for inactivated HRP in strong alkali solution. The cyclic voltammetry results indicate that the adsorbed native HRP can directly catalyse the reduction of hydrogen peroxide, demonstrating that a direct electron transfer reduction occurred between the enzyme and HOPG electrode, whereas the corresponding cyclic voltammograms for denatured HRP and inactivated HRP adsorbed on HOPG electrodes indicate a lack of ability to catalyse H2O2 reduction, which confirms that the HRP molecules lost their biological activity. Obviously, electrochemical results powerfully support in situ STM observations.

AN ORGANIC-PHASE ENZYME ELECTRODE BASED ON AN APPARENT DIRECT ELECTRON-TRANSFER BETWEEN A GRAPHITE ELECTRODE AND IMMOBILIZED HORSERADISH-PEROXIDASE

A mediatorless horseradish peroxidase (HRP) enzyme electrode operated in nonaqueous media is constructed by cryohydrogel immobilization.

REDOX REACTION OF PEROXIDASE AT POLY(O-PHENYLENEDIAMINE) MODIFIED ELECTRODE

The redox conversion of heme-containing protein horseradish peroxidase (HRP), which has a molar mass of 40,000, was studied. The conversion was obtained at an electrochemical polymerized o-phenylenediamine (PPD) film-modified platinum electrode. Optical c

MEDIATORLESS HYDROGEN-PEROXIDE ELECTRODE BASED ON HORSERADISH-PEROXIDASE ENTRAPPED IN POLY(O-PHENYLENEDIAMINE)

A mediatorless H2O2 sensor based on coelectropolymerization of horse radish peroxidase (HRP) and o-phenylenediamine (o-PD) is described. The electrode responds to H2O2 in a few seconds and gives a current density of 73.3 nA 1 mu mol(-1) cm(-2) at -100 mV

cDNA cloning and mRNA expression of a selenium-dependent glutathione peroxidase from Zhikong scallop Chlamys farreri

The glutathione peroxidases are essential enzymes of the cellular antioxidant defence system. In the present study, the full-length cDNA sequence encoding an extracellular glutathione peroxidase (designated CfGPx3) was isolated from Zhikong scallop Chlamys farreri. The complete cDNA was of 1194 bp, containing a 5' untranslated region (UTR) of 50 bp, a 3' UTR of 490 bp and an open reading frame (ORF) of 654 bp encoding a polypeptide of 217 amino acids. CfGPx3 possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase, such as the selenocysteine encoded by stop codon UGA, the GPx signature motif ((96)LGVPCNQFI(103)) and the active site motif ((WNFEKF184)-W-179). The high similarity of CfGPx3 with GPx from other organisms indicated that CfGPx3 should be a new member of the glutathione peroxidase family. By fluorescent quantitative real-time PCR, the CfGPx3 mRNA was universally detected in the tissues of haemocytes, gill, gonad, muscle and hepatopancreas with the highest expression in hepatopancreas. After scallops were challenged by Listonella anguillarum, the expression level of CfGPx3 transcript in haemocytes was significantly up-regulated (P<0.05) at 8 h post challenge. These results suggested that CfGPx3 was potentially involved in the immune response of scallops and perhaps contributed to the protective effects against oxidative stress. (C) 2010 Elsevier Inc. All rights reserved.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis:in vitro evidence in cultured epithelial cells

BACKGROUND:
Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR.
OBJECTIVES:
The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects.
METHODS:
Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function.
RESULTS:
Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway.
CONCLUSIONS:
This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.

A minor catalase/peroxidase from Burkholderia cenocepacia is required for normal aconitase activity

The opportunistic bacterium Burkholderia cenocepacia C5424 contains two catalase/peroxidase genes, katA and katB. To investigate the functions of these genes, katA and katB mutants were generated by targeted integration of suicide plasmids into the katA and katB genes. The catalase/peroxidase activity of the katA mutant was not affected as compared with that of the parental strain, while no catalase/peroxidase activity was detected in the katB mutant. However, the katA mutant displayed reduced resistance to hydrogen peroxide under iron limitation, while the katB mutant showed hypersensitivity to hydrogen peroxide, and reduced growth under all conditions tested. The katA mutant displayed reduced growth only in the presence of carbon sources that are metabolized through the tricarboxylic acid (TCA) cycle, as the growth defect was abrogated in cultures supplemented with glucose or glycerol. This phenotype was also correlated with a marked reduction in aconitase activity. In contrast, aconitase activity was not reduced in the katB mutant and parental strains. The authors conclude that the KatA protein is a specialized catalase/peroxidase that has a novel function by contributing to maintain the normal activity of the TCA cycle, while KatB is a classical catalase/peroxidase that plays a global role in cellular protection against oxidative stress.

Plasma glutathione peroxidase activity is reduced in haemodialysis patients

Cardiovascular disease is the major cause of morbidity and mortality in patients with end-stage renal failure. Increased free radical production and antioxidant depletion may contribute to the greatly increased risk of atherosclerosis in these patients. Glutathione peroxidase (GPX) is an important antioxidant, the plasma form of which is synthesized mainly in the kidney (eGPX). The aim of this study was to assess the activity of eGPX in patients with end-stage renal failure on haemodialysis. Venous blood was collected from 87 haemodialysis patients immediately prior to and after dialysis and from 70 healthy controls. Serum eGPX activity was measured using hydrogen peroxide as substrate and immunoreactivity determined by ELISA. eGPX activity was significantly reduced in dialysis patients when compared to controls (106 +/- 2.7 and 281 +/- 3.6 U/l respectively, p <0.001). Following haemodialysis, eGPX activity rose significantly to 146 +/- 3.8 U/l, p <0.001, although remaining below control values (p <0.005). Immunoreactive eGPX, however, was similar in all groups (pre-dialysis 14.10 +/- 1.26 microg/ml, post-dialysis 14.58 +/- 1.35 microg/ml, controls 15.20 +/- 1.62 microg/ml, p = NS). A decrease was observed in the specific activity of eGPX in patients when compared to controls (8.81 +/- 1.14, 10.71 +/- 1.54 and 21.97 +/- 1.68 U/mg respectively, p <0.0001). eGPX activity is impaired in patients undergoing haemodialysis and so may contribute to atherogenesis in renal failure.

Glutathione peroxidase 1 genotype is associated with an increased risk of coronary artery disease

Objective Oxidative stress is implicated in the pathogenesis of many human diseases including atherosclerosis. Human glutathione peroxidase 1 (hgpx1) participates in limiting cellular damage caused by oxidation. A characteristic polyalanine sequence polymorphism in exon 1 of hgpx1 produces three alleles with five, six or seven alanine (ALA) repeats in this sequence. The objective of this study was to determine whether hgpx1 genotype is associated with an altered risk of coronary artery disease (CAD).

Methods The frequency of the ALA6 allele was determined in 207 men with angiographic evidence of significant CAD compared to a control group (n = 146), by analysing the lengths of polymerase chain reaction fragments containing the ALA repeat polymorphism. Additional information was collected on severity of CAD, presence or absence of a prior acute myocardial infarction (AMI), smoking status, body mass index (BMI) and other clinical data.

Results There was a significant association between individuals with at least one ALA6 allele and an increased risk of CAD after adjustment for age, BMI and smoking status (odds ratio, 2.07, 95% confidence interval, 1.08-3.99, P = 0.029). However, there was no association between hgpx1 genotype and a previous history of AMI or hgpx1 genotype and severity of CAD.

Conclusion We conclude that individuals possessing one or two ALA6 alleles appear to be at a modest increased risk of CAD. This observation merits further investigation in other patient populations.