A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots

Direct electron transfer of hemoglobin modified with quantum dots (QDs) (CdS) has been performed at a normal graphite electrode. The response current is linearly dependent on the scan rate, indicating the direct electrochemistry of hemoglobin in that case is a surface-controlled electrode process. UV–vis spectra suggest that the conformation of hemoglobin modified with CdS is little different from that of hemoglobin alone, and the conformation changes reversibly in the pH range 3.0–10.0. The hemoglobin in a QD film can retain its bioactivity and the modified electrode can work as a hydrogen peroxide biosensor because of its peroxidase-like activity. This biosensor shows an excellent response to the reduction of H₂O₂ without the aid of an electron mediator. The catalytic current shows a linear dependence on the concentration of H₂O₂ in the range 5 × 10⁻⁷–3 × 10⁻⁴ M with a detection limit of 6 × 10⁻⁸ M. The response shows Michaelis–Menten behavior at higher H₂O₂ concentrations and the apparent Michaelis–Menten constant is estimated to be 112 μM.     

Direct electrochemistry and electrocatalytic activity of catalase incorporated onto multiwall carbon nanotubes-modified glassy carbon electrode

The direct voltammetry and electrocatalytic properties of catalase, which was adsorbed on the surface of multiwall carbon nanotubes (MWCNTs), was investigated. A pair of well-defined and nearly reversible cyclic voltammetry peaks for Fe(III)/Fe(II) redox couple of catalase adsorbed on the surface of MWCNTs at approximately -0.05 V versus reference electrode in pH 6.5 buffer solution, indicating the direct electron transfer between catalase and electrode. The surface coverage of catalase immobilized on MWCNTs glassy carbon electrode was approximately 2.4x10(-10) molcm-2. The transfer coefficient (alpha) was calculated to be 0.4, and the heterogeneous electron transfer rate constant was 80 s-1 in pH 7, indicating great facilitation of the electron transfer between catalase and MWCNTs adsorbed on the electrode surface. The formal potential of catalase Fe(III)/Fe(II) couple in MWCNTs film had a linear relationship with pH values between 2 and 11 with a slope of 58 mV/pH, showing that the electron transfer is accompanied by single proton transportation. Catalase adsorbed on MWCNTs exhibits a remarkable electrocatalytic activity toward the reduction of oxygen and hydrogen peroxide. The value for calculated Michaelis-Menten constant (1.70 mM) was high, indicating the potential applicability of the films as a new type of reagentless biosensor based on the direct electrochemistry of the catalase enzyme.     

Development of an oxygen-rich biosensor using enzymatic reaction

This work reports a novel strategy for the development of an O₂-rich biosensor. The principle is based on an enzymatic reaction between catalase and H₂O₂ to release O₂, thus to increase the O₂ amount in the enzyme matrix. This method improves the determination reliability by alleviating the O₂ dependence.     

Possible role of catalase in post-dormancy bud break in grapevines

Changes in the activity of catalase (Cat) and in the levels of H2O2 were followed throughout dormancy in buds of grapevines (Vitis vinifera L.). In grapevines grown in the Elqui valley in Chile, a region with warm-winters, the activity of Cat increased during the recess period of buds, reaching a maximum and thereafter decreased to less than one third of its maximal activity. Three isoforms of Cat were detected in extracts of buds by native PAGE analysis, and the extracted activity was inhibited competitively by hydrogen cyanamide (HC), a potent bud-break agent. Furthermore, HC applications to field-grown grapevines in addition to the expected effect on advancing bud break, reduced the Cat activity during bud dormancy. Similar reductions were observed during dormancy in buds of grapevines grown in the Central valley in Chile, a region with temperate winters, suggesting that HC and winter chilling inhibits the activity of the main H2O2 degrading enzyme in grape buds. A transient rise in H2O2 levels preceded the release of buds from endodormancy, moreover, the peak of H2O2 and the onset of bud break occurred earlier in HC treated than in control grapevines, suggesting the participation of H2O2 as a signal molecule in the release of endodormancy in grape buds. The relationship between Cat inhibition, rise in H2O2 levels and initiation of bud break are discussed.     

Determination of the activity of catalase using a europium(III)-tetracycline-derived fluorescent substrate

A one-step method is described for the fluorometric determination of the activity of the enzyme catalase (EC 1.11.1.6.), based on the finding that H(2)O(2) in the europium (III)-tetracycline-hydrogen peroxide system is consumed by catalase. This is accompanied by a large decrease in both fluorescence intensity and decay time. The limit of detection (LOD; at S/N=3) for catalase at 30 degrees C for a 10-min kinetic assay is 1.0 unit/mL, with a linear range from 1.0 to 10 unit/mL. At an incubation time of 30 min at 37 degrees C for a one-point assay, the LOD is 0.046 unit/mL, with a linear range from 46 to 400 munit/mL. The assay was performed on microtiterplates and is fully compatible with existing plate readers. It is a one-step, simple, and sensitive method suitable for both continuous kinetic and one-point detections, does not require the addition of other substrates, and works best at neutral pH (with an optimum at pH 6.9). The reagent has the typical spectral features of a europium-ligand complex including a large Stokes shift (210 nm), a red line-like emission (centered at 616 nm), and a decay time in the microsecond domain. It is also the first europium-based probe that is compatible with the 405-nm diode laser. In summary, the new assay provides distinct advantages over direct ultraviolet detection and over the two-reagent (peroxidase) method.     

Activity of magnetite-immobilized catalase in hydrogen peroxide decomposition

The present work analyzes the activity in decomposition of H₂O₂ using magnetite-immobilized catalase. The support of catalase is a glutaraldehyde-treated magnetite (Fe₃O₄). The data obtained in the H₂O₂ decomposition are analyzed. The fitting of the initial rate of the H₂O₂ decomposition versus hydrogen peroxide concentration data is discussed using a specific program for enzyme kinetics modeling (Leonora). The free catalase from Aspergillus niger (3.5 or 10 U/mL) does not show substrate inactivation up to 0.4 M H₂O₂. The immobilized catalase at low catalyst concentration shows substrate inhibition. Using 1 mg/mL of supported catalase the predicted maximum activity is higher than in the case of the free catalase at similar catalase concentration, although the optimum temperature is lower (40 °C versus 60 °C).     

Graphitized macroporous carbon microarray with hierarchical mesopores as host for the fabrication of electrochemical biosensor

A novel graphitized ordered macroporous carbon (GMC, pore size 380 nm) with hierarchical mesopores (2–30 nm) and high graphitization degree was prepared by nickel-catalyzed graphitization of polystyrene arrays. The obtained GMC possessed high specific surface area, large pore volume, and good electrical conductivity, which was explored for the enzyme entrapment and biosensor fabrication by a facile method. With advantages of novel nanostructure and good electrical conductivity, direct electrochemistry of hemoglobin (a model protein) was observed on the GMC-based biocomposite with a formal potential of −0.36 V (vs. Ag/AgCl) and an apparent heterogeneous electron transfer rate constant (k_s) of 1.2 s⁻¹ in pH 7.0 buffer. Comparative studies revealed that GMC offered significant advantages over carbon nanotubes (CNTs) in facilitating direct electron transfer of entrapped Hb. The fabricated biosensor exhibited good sensitivity (101.6 mA cm⁻² M⁻¹) and reproducibility, wide linear range (1–267 μM), low detection limit (0.1 μM), and good long-term stability for H₂O₂ detection. GMC proved to be a promising matrix for enzyme entrapment and biosensor fabrication, and may find wide potential applications in biomedical detection and environmental analyses.     

Serratia marcescens SYBC08 catalase isolated from sludge containing hydrogen peroxide shows increased catalase production by regulation of carbon metabolism

A high‐catalase‐producing strain, which was isolated from sludge containing hydrogen peroxide, was identified as Serratia marcescens SYBC08 by 16S rDNA sequence analysis. Serratia spp. was reported as non‐spore‐forming bacterium (except S. marcescens spp. sakuensis), but in our study electron microscopic observation revealed that the strain did produce spores. The content of the main fatty acid C_16:0 (14.8%) was significantly different from that of S. marcescens spp. sakuensis (33.2%) and S. marcescens spp. marcescens DSM 30121^T (34.8%), and the biochemical characteristics were not identical to those of S. marcescens spp. sakuensis. We speculate that the relatively high catalase activity and the spore structures may enable the strain to survive in a hydrogen peroxide environment. The most suitable carbon and nitrogen sources for the catalase production by S. marcescens SYBC08 were citric acid and corn steep liquor powder. A strategy of carbon metabolism regulation to enhance the catalase production was exploited. In the 7‐L fermenter, catalase production (20 353 U/mL) obtained in the presence of glucose and citric acid was 1.68‐ and 1.31‐fold higher than that obtained in the presence of glucose or citric acid, at equimolar carbon concentration. This production yield was much higher than that of many catalase‐producing strains, but only slightly lower than the production by Micrococcus luteus (34 601 U/mL). The results suggest that the new spore‐forming S. marcescens SYBC08 is a potential candidate for the production of catalase.     

Study and improvement of the conditions for production of a novel alkali stable catalase

Catalase (CAT) is an enzyme capable of catalyzing the conversion of H(2)O(2) to O(2) and H(2)O. It has recently acquired interest due to its attractive potential application in the textile industries. In a previous study, a bacterium with slight halophilic and alkaliphilic characteristics, Bacillus sp. F26, was isolated and found to produce high-level alkaline CAT. In the present study, the effects of culture conditions on the CAT production were investigated. The results showed that the highest activity of CAT (13.9 U/mg protein) was obtained when glucose (15 g/L) was used as carbon source. The utilization of the mixture of corn steep liquid and beef extract stimulated both bacterial growth and CAT synthesis. The highest biomass (4.5 g/L) and activity of CAT (16.5 U/mg protein) were found synchronously when 10 g/L corn steep liquid and 10 g/L beef extract were used as nitrogen source. The addition of H(2)O(2) as an oxidative stress was used to enhance CAT production in the flasks. It was found that the activity of CAT was increased by 51.3-22.8 U/mg protein compared with the control when 2 mmol/L H(2)O(2) was added at later exponential phases (16 h), although the cell growth was significantly inhibited. Based on the above, an exponential H(2)O(2) feed strategy was developed, in which the feed rate of H(2)O(2) was controlled according to specific cell growth rate (mu). In this way, the maximum CAT production (29.9 U/mL) was obtained, which was 92.8 and 20.7% higher than that in batch and constant rate fed-batch fermentation, respectively.     

Haemonchus contortus utilises catalase in defence against exogenous hydrogen peroxide in vitro

The toxicity of activated oxygen species towards adult Haemonchus contortus nematodes was examined in in vitro assays using ingestion of [³H]inulin to assess nematode viability. Both glucose/glucose oxidase (generation of hydrogen peroxide) and xanthine/xanthine oxidase (generation of superoxide anion) systems showed concentration-dependant toxicity to the nematodes. Both adult and larval Haemonchus contortus enzyme preparations showed significant catalase activities. Adult nematodes exposed to aminotriazole for 24 h showed catalase activities reduced to less than 20% of controls. Aminotriazole-treated nematodes exposed to a glucose/glucose oxidase system were significantly more susceptible to the toxic effects of the oxidant-generating system than controls (no aminotriazole pre-treatment). The concentration of glucose oxidase required to inhibit feeding by 50% was decreased 33-fold in aminotriazole-treated nematodes compared with controls. The effect of aminotriazole pre-treatment implicates hydrogen peroxide as a significant toxic agent in the glucose/glucose oxidase system. It is apparent that inhibition of Haemonchus contortus catalase increases the susceptibility of the parasite to the toxic effects of hydrogen peroxide, demonstrating a protective role for this enzyme. This suggests that catalase has the potential to play a significant role in the defence of this parasite against hydrogen peroxide produced as part of the respiratory burst of activated phagocytes within the host during its response to nematode infection.     

Novel 3-dimensional bioelectrode for mediatorless bioelectrochemical denitrification

A novel bioelectrochemical method for denitrification was developed using electricity as the electron donor. The novel electrode contained both Ochrobactrum anthropi SY509, which was permeabilized as a biocatalyst, and copper powder as a conducting material. Using this electrode, a high denitrification efficiency of 1 mmol N-NO (3) (-) /g dry cell.h was achieved via direct electron transfer without using mediator.     

Anomalous phylogenies based on bacterial catalase gene sequences

Phylogenies based on nine prokaryotic catalase sequences demonstrate no relationship to phylogenies based on rDNA sequences or other known criteria. When this observation is considered together with the monophyletic relationship observed for eukaryotic catalase sequences, it seems likely that the catalase gene sequence has migrated repeatedly from eukaryotes to prokaryotes.     

Mechanisms of catalase activity of heme peroxidases

In the absence of exogenous electron donors monofunctional heme peroxidases can slowly degrade hydrogen peroxide following a mechanism different from monofunctional catalases. This pseudo-catalase cycle involves several redox intermediates including Compounds I, II and III, hydrogen peroxide reduction and oxidation reactions as well as release of both dioxygen and superoxide. The rate of decay of oxyferrous complex determines the rate-limiting step and the enzymes’ resistance to inactivation. Homologous bifunctional catalase-peroxidases (KatGs) are unique in having both a peroxidase and high hydrogen dismutation activity without inhibition reactions. It is demonstrated that KatGs follow a similar reaction pathway as monofunctional peroxidases, but use a unique post-translational distal modification (Met⁺-Tyr-Trp adduct) in close vicinity to the heme as radical site that enhances turnover of oxyferrous heme and avoids release of superoxide. Similarities and differences between monofunctional peroxidases and bifunctional KatGs are discussed and mechanisms of pseudo-catalase activity are proposed.     

Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

JNK1 activity lowers the cellular production of H2O2 and modulates the growth arrest response to scavenging of H2O2 by catalase

Hydrogen peroxide (H(2)O(2)) can interact with intracellular signaling pathways to regulate cell behavior. The c-Jun NH(2)-terminal kinase 1 (JNK1) signal, involved in diverse aspects of cellular functioning, is implicated as a cell sensor of redox stress. The growth-inhibitory effect of both high-level H(2)O(2) and H(2)O(2)-scavenging catalase treatments is accompanied by increased JNK1 activity. To investigate the role of this response in growth regulation, the JNK1 signal was increased by the introduction of ectopic HA-JNK1. HA-JNK1 expression correlated with increases in basal c-Jun phosphorylation in a dose-dependent manner. Transient expression of HA-JNK1 potentiated cell growth arrest by catalase; however, with stable expression a degree of resistance to this response was observed. Resistance was accompanied by a lowered endogenous production of H(2)O(2). Transient HA-JNK1 expression also reduced H(2)O(2) generation, and this effect was reversed by the JNK inhibitor SP600125. These results indicate that the JNK1 stress response contributes to growth inhibition by catalase treatment via inhibition of cellular H(2)O(2) production. Stable amplification of the JNK1 pathway leads to cellular adaptation to its signal, resulting in a diminished reliance upon H(2)O(2) for efficient growth.     

Direct electrochemistry of cytochrome c at ordered macroporous active carbon electrode

Three-dimensionally (3D) ordered macroporous active carbon has been fabricated and used as electrode substrate for the direct electrochemistry of horse heart cytochrome c (Cyt c). The Cyt c immobilized on the surface of the ordered macroporous active carbon shows a pair of well-defined and nearly reversible redox waves at the formal potential of −0.033 V in pH 6.8 phosphate buffer solution. The interaction between Cyt c and the 3D macroporous active carbon makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods have been used to investigate the interaction between Cyt c and the porous active carbon. The immobilized Cyt c maintains its biological activity, and shows a surface controlled electrode process with the electron-transfer rate constant (k_s) of 17.6 s⁻¹ and the charge-transfer coefficient (a) of 0.52, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). A potential application of the Cyt c-immobilized porous carbon electrode as a biosensor to monitor H₂O₂ has been investigated. The steady-state current response increases linearly with H₂O₂ concentration from 2.0 × 10⁻⁵ to 2.4 × 10⁻⁴ mol l⁻¹. The detection limit (3σ) for determination of H₂O₂ has been found to be 1.46 × 10⁻⁵ mol l⁻¹.     

Caspase-8 dependent trail-induced apoptosis in cancer cell lines is inhibited by vitamin C and catalase

TNF-related apoptosis-inducing ligand (TRAIL/ Apo-2L) is a member of the TNF family of apoptosis-inducing proteins that initiates apoptosis in a variety of neoplastic cells while displaying minimal or absent cytotoxicity to most normal cells. Therefore, TRAIL is currently considered a promising target to develop anti-cancer therapies. TRAIL-receptor ligation recruits and activates pro-caspase-8, which in turn activates proteins that mediate disruption of the mitochondrial membranes. These events lead to the nuclear and cytosolic damage characteristic of apoptosis. Here we report that TRAIL-induced apoptosis is mediated by oxidative stress and that vitamin C (ascorbic acid), a potent nutritional antioxidant, protects cancer cell lines from apoptosis induced by TRAIL. Vitamin C impedes the elevation of reactive oxygen species (ROS) levels induced by TRAIL and impairs caspase-8 activation. We found that the removal of hydrogen peroxide by extracellular catalase during TRAIL-induced apoptosis also impairs caspase-8 activation. These data suggest that hydrogen peroxide is produced during TRAIL-receptor ligation, and that the increase of intracellular ROS regulates the activation of caspase-8 during apoptosis. Additionally we propose a mechanism by which cancer cells might resist apoptosis via TRAIL, by the intake of the nutritional antioxidant vitamin C. This work was supported by grants from the National Institutes of Health (CA 30388), the New York State Department of Health (M020113) and the Lebensfeld Foundation.