Activity Of Horseradish Peroxidase Research Articles

DNA template-assisted modulation of horseradish peroxidase activity

The precise alignment of DNA molecules by Watson–Crick base-pairing combined with its polymeric characteristics have allowed DNA to be used as a template or scaffold for assembling materials. In this work, we investigate the role of calf-thymus DNA as a template for enhancing the horseradish peroxidase (HRP)-mediated oxidation of phenol and phenolic derivatives. The HRP-catalyzed oxidation of phenol into polyphenolic products and in presence of 4-aminoantipyrine into quinoneimine dye complexes is studied. Visible spectroscopy reveals an increased yield of both products of the enzymatic reaction in presence of calf-thymus DNA and is attributed to the prearrangement of the corresponding substrates on the DNA. The concentrations of calf-thymus DNA and the substrates are found to affect the nature of prearrangement and subsequent formation of polymeric or co-oxidation products. Also, phenolic derivatives with different aromatic substitutions display divergent propensities towards product formation in presence of the DNA template. Our results demonstrate the ability of calf-thymus DNA to modulate the activity of HRP and exercise control on the nature of products formed. This work highlights the potential of using DNA as a template for influencing enzymatic reactions involving aromatic substrates.

Preparation of water-dispersible poly[aniline-co-sodium N-(1-one-butyric acid) aniline]–zinc oxide nanocomposite for utilization in an electrochemical sensor

An electrochemical sensor based on an enzymatic poly[aniline-co-sodium N-(1-one-butyric acid) aniline]–zinc oxide (SPAnNa–ZnO) nanocomposite-modified gold electrode was developed for the determination of hydrogen peroxide (H2O2). The SPAnNa–ZnO nanocomposites dispersed stably in an aqueous solution because the small quantity of polyelectrolytic SPAnNa on the ZnO surface prevented nanocomposite aggregation. In addition, the electroactivity and electrochemical behavior of SPAnNa were enhanced in phosphate buffer solution at pH 7.0 by hybridization with spherical ZnO nanoparticles. These negatively charged SPAnNa–ZnO nanocomposites provided a favorable environment for the immobilization of horseradish peroxidase (HRP) by electrostatic interactions. The load quantity of immobilized HRP was approximately 15 wt% on the SPAnNa–ZnO nanocomposite. This non-destructive immobilization maintained the activity of HRP, thereby enabling effective H2O2 detection. The sensor exhibited a linear detection range from 10 μM to 2 mM with a relatively high sensitivity of 519.8 μA mM−1 cm−2 at an applied potential of −0.4 V.

Oxidation activity of horseradish peroxidase hosted in molecular sieves: spectroscopic investigations show hindering of the enzyme activity

Horseradish peroxidase immobilization inside molecular sieves with pores hexagonally structured as such or functionalized with copper ions is presented in comparison with the bidimensional immobilization onto aerosil A380 particles. Immobilization was performed under mild conditions that do not lead to enzyme denaturation. Guest–host interactions were pursued spectrophotometrically and by thermal analysis measurements. FTIR spectroscopy showed no major secondary structural change for the entrapped enzyme despite some influence of the support functionalization that can be supposed. The activity of the immobilized enzyme was tested in the oxidation of Alizarin Red S (ARS) with hydrogen peroxide. It was found that the dye oxidation is catalyzed by the enzyme in homogeneous reaction but the rate becomes much slower in heterogeneous catalytic processes with prepared catalysts. It seems that horseradish peroxidase immobilization in simple porous silica or alumino-silica post synthesis blocks the enzyme activity. In addition, copper ions make a stable complex with ARS hindering its oxidation as well.

Nanomolar Hydrogen Peroxide Detection Using Horseradish Peroxidase Covalently Linked to Undoped Nanocrystalline Diamond Surfaces

In this article, we report on the low-level detection of hydrogen peroxide, a key player in the redox signaling pathway and a toxic product in the cellular system, using a colorimetric solution assay. Amine-terminated undoped nanocrystalline diamond thin films were grown on glass using a linear-antenna microwave plasma CVD process. The diamond surface consists mainly of -NH(2) termination. The aminated diamond surface was decorated with horseradish peroxidase (HRP) enzyme using carbodiimide coupling chemistry. The success of the HRP immobilization was confirmed by X-ray photoelectron spectroscopy (XPS). The enzymatic activity of immobilized HRP was determined with a colorimetric test based on the HRP-catalyzed oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sufonic acid (ABTS) in the presence of hydrogen peroxide. The surface coverage of active HRP was estimated to be Γ = 7.3 × 10(13) molecules cm(-2). The use of the functionalized diamond surface as an optical sensor for the detection of hydrogen peroxide with a detection limit of 35 nM was demonstrated.

Enhanced enzyme activity through electron transfer between single-walled carbon nanotubes and horseradish peroxidase

Better understanding of electron transfer (ET) taking place at the nano-bio interface can guide design of more effective functional materials used in fuel cells, biosensors, and medical devices. Single-walled carbon nanotube (SWCNT) coupled with biological enzymes serves as a model system for studying the ET mechanism, as demonstrated in the present study. SWCNT enhanced the activity of horseradish peroxidase (HRP) in the solution-based redox reaction by binding to HRP at a site proximate to the enzyme’s activity center and participating in the ET process. ET to and from SWCNT was clearly observable using near-infrared spectroscopy. The capability of SWCNT in receiving electrons and the direct attachment of HRP to the surface of SWCNT strongly affected the enzyme activity, further confirming the involvement of SWCNT in ET.

Peroxidase-mediated dealkylation of tamoxifen, detected by electrospray ionization–mass spectrometry, and activation to form DNA adducts

Tamoxifen (TAM) is extensively used for the treatment and prevention of breast cancer. Associated with TAM treatment is a two- to eightfold increase in risk of endometrial cancer. To understand the mechanisms associated with this increased risk several pathways for TAM metabolism and DNA adduct formation have been studied. The purpose of this study was to investigate the role of peroxidase enzymes in the metabolism of TAM and its activation to form DNA adducts. Using advanced tandem mass spectrometry we have investigated the peroxidase-mediated metabolism of TAM. Incubation of TAM with horseradish peroxidase (HRP) and H 2O 2 produced multiple metabolites. Electrospray ionization–MS/MS analysis of the metabolites demonstrated a peak at 301.3 m/z with daughter ions at 183.0, 166.9, 128.9, and 120.9 m/z, which identified the metabolite as metabolite E (ME). The levels of ME were significantly inhibited by the addition of ascorbic acid to the incubation mixture. Co-incubation of either TAM or ME and DNA with HRP and H 2O 2 produced three DNA adducts with a RAL of 1.97 ± 0.01 × 10 − 7 and 8.45 ± 2.7 × 10 − 7 . Oxidation of ME with MnO 2 produced metabolite E quinone methide (MEQM). Furthermore, incubation of either TAM or ME with HRP and H 2O 2 resulted in formation of MEQM. Reaction of calf thymus DNA with MEQM produced three DNA adducts with a RAL of 9.8 ± 1.0 × 10 − 7 . Rechromatography analyses indicated that DNA adducts 1, 2, and 3 formed in the HRP activation of either TAM or ME were the same as those formed by the chemical reaction of DNA with MEQM. The results of these studies demonstrate that peroxidase enzymes can both metabolize TAM to form the primary metabolite ME and activate ME to a quinone methide intermediate, which reacts with DNA to form adducts. It is possible that peroxidase enzymes or peroxidase-like activity in endometrium could contribute to the formation of DNA damage and genotoxic effects in endometrium after TAM administration.

THE EFFECT OF PULSED MAGNETIC FIELD ON HORSERADISH PEROXIDASE

ABSTRACTThe effect of pulsed magnetic field (PMF) on the activity and structure of horseradish peroxidase (HRP) in buffer solution was investigated. HRP was inactivated when exposed to PMF at field strengths of 2.0, 3.0 and 4.0 tesla (T) for 5 to 40 pulses. The maximum inactivation of HRP was achieved at 4.0 T for 40 pulses, in which 67.67% of the enzyme activity was lost. Further experiments showed that the activity of HRP was not significantly affected by either storage time at 4C or the heat produced during lower intensity PMF (from 1.0 to 3.0 T). The inactivation data were adjusted to the Bigelow, Hülsheger and Weibull models. Results showed that the Weibull model successfully described the relationship between residual activity of HRP and pulse strength as well as pulse number. Ultraviolet and intrinsic fluorescence spectra analysis proved that the PMF‐induced inactivation correlated with alteration of the tertiary structure of HRP.PRACTICAL APPLICATIONSAs a kind of enzyme present in many foods, when peroxidase (POD) is exposed to air, it will catalyze some reactions which have a negative effect on sensorial and nutritional qualities of the food. In this article, pulsed magnetic field was proved to be a potential processing method to inactivate POD without destroying the sensorial and nutritional qualities. The kinetic models also offer some parameters for designing methods to prolong shelf life of agricultural products.

A Third‐Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized in Carbon Nanotubes/ SBA‐15 Film

AbstractA new third‐generation biosensor for H2O2 assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of carbon nanotubes (CNTs)‐SBA‐15 modified gold electrode. The biological activity of HRP immobilizing in the composite film was characterized by UV‐vis spectra. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H2O2. The effects of the experimental variables such as solution pH and working potential were investigated using steady‐state amperometry. Under the optimal conditions, the resulting biosensor showed a linear range from 1 µM to 7 mM and a detection limit of 0.5 µM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.

Overcoming the adverse effects of crosslinking in biosensors via addition of PEG: Improved sensing of hydrogen peroxide using immobilized peroxidase

Glutaraldehyde (GA) is widely used as a crosslinker to immobilize enzymes, for examples in biosensors, but often causes partial denaturation. We find that the proper use of poly(ethylene glycol) (PEG) during the crosslinking process can fully preserve the native state and activity of horseradish peroxidase (HRP). An amperometric biosensor was developed based on these findings for the direct determination of hydrogen peroxide. UV-Vis and FTIR spectroscopy reveal that the HRP entrapped in a polypyrrole matrix retains its native structure. The addition of PEG increases the sensitivity and stability of the biosensor and prevents many of effects caused by intra-crosslinking via GA. The biosensor was operated at a potential of −350 mV (vs Ag/AgCl) without any mediator and gave a linear response to H2O2 in the 5 to 190 μM concentration range. The apparent Michaelis-Menten constant is 3.37 mM, and maximal current is as high as 3.43 μA. The surface of the biosensor was characterized by atomic force microscopy operated in the tapping mode.

Determination of Selected Heavy Metals Using Amperometric Horseradish Peroxidase (HRP) Inhibition Biosensor

Fabrication of an amperometric biosensor for determination of heavy metals in tap water based on the inhibition of horseradish peroxidase activity is reported. The inhibition of the biosensor's response to 0.95 mM H2O2 at −200 mV (vs. Ag/AgCl) followed the order, Cd2+ < Cu2+ < Pb2+. The detection limits were 0.09 ppb, 0.03 ppb, and 0.10 ppb for Cd2+, Pb2+, and Cu2+, respectively. Kinetic parameters (apparent Michaelis-Menten constant) and Imax (maximum current) obtained in the absence ( = 1.16 mM, Imax = 0.51 µA) and the presence ( = 1.13 mM, Imax = 0.36) of 3.13 ppm Cd2+ showed that the enzyme inhibition process was reversible and non-competitive, and the corresponding KI (inhibition constant) value was 0.026 mM. After two weeks, the biosensor retained >95% of its initial response and gave a reproducibility of 3.8% and precision of 2.1%.

Development of two highly sensitive immunoassays for detection of copper ions and a suite of relevant immunochemicals

Availability of highly sensitive assays for metal ions can help monitor and manage the environmental and food contamination. In the present study, a monoclonal antibody against Copper(II)–ethylenediaminetetraacetic acid was used to develop two sensitive ELISAs for Cu(II) analysis. Cobalt(II)–EDTA–BSA was the coating antigen in a heterologous indirect competitive ELISA (hicELISA), whereas Co(II)–EDTA–BSA–horseradish peroxidase (HRP) was the enzyme tracer in a heterologous direct competitive ELISA (hdcELISA). Both ELISAs were validated for detecting the content of Cu(II) in environmental waters. The ELISA data agreed well with those from graphite furnace atomic absorption spectroscopy. The methods of developing the Cu(II) hicELISA and hdcELISA are potentially applicable for developing ELISAs for other metals. The chelator–protein complexes such as EDTA–BSA and EDTA–BSA–HRP can form a suite of metal complexes having the consistent hapten density, location and orientation on the conjugates except the difference of the metal core, which can be used as ideal reagents to investigate the relationship between assay sensitivity and antibody affinities for the haptens and the analytes. The strategy of conjugating a haptenated protein directly with HRP can reduce the loss of HRP activity during the conjugation reaction and thus can be applicable for the development of ELISAs for small molecules.

Photoswitching of Enzyme Activity of Horseradish Peroxidase Intercalated into Semiconducting Layers

The present study demonstrates that enzymatic reaction of horseradish peroxidase (HRP) inserted into semiconducting iron-doped titanate (FT) layers can be controlled by ultraviolet (UV) light irrad...

Effect of the structure of imidazolium cations in [BF4]−-type ionic liquids on direct electrochemistry and electrocatalysis of horseradish peroxidase in Nafion films

The direct electrochemistry and bioelectrocatalysis of horseradish peroxidase (HRP) in Nafion films at glassy carbon electrode (GCE) was investigated in three [BF(4)](-)-type room-temperature ionic liquids (ILs) to understand the structural effect of imidazolium cations. The three ILs are 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim][BF(4)]), 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]) and 1-hexyl-3-methylimidazolium tetrafluoroborate ([Hmim][BF(4)]). A small amount of water in the three ILs is indispensable for maintaining the electrochemical activity of HRP in Nafion films, and the optimum water contents decrease with the increase of alkyl chain length on imidazole ring. Analysis shows that the optimum water contents are primarily determined by the hydrophilicity of ILs used. In contrast to aqueous medium, ILs media facilitate the direct electron transfer of HRP, and the electrochemical parameters obtained in different ILs are obviously related to the nature of ILs. The direct electron transfer between HRP and GCE is a surface-confined quasi-reversible single electron transfer process. The apparent heterogeneous electron transfer rate constant decreases gradually with the increase of alkyl chain length on imidazole ring, but the changing extent is relatively small. The electrocatalytic reduction current of H(2)O(2) at the present electrode decreases obviously with the increase of alkyl chain length, and the mass transfer of H(2)O(2) via diffusion in ILs should be responsible for the change. In addition, the modified electrode has good stability and reproducibility; the ability to tolerate high levels of F(-) has been greatly enhanced due to the use of Nafion film. When an appropriate mediator is included in the sensing layer, a sensitive nonaqueous biosensor could be fabricated.

Study of High Voltage Electric Field on the Mechanism of Horseradish Peroxidase Activity

The activity of Horseradish Peroxidase (HRP) was enhanced with different strength of high voltage electric field. The results have shown that Ultraviolet-Visible spectra have been changed obviously. Experimental results indicate that the conformation of HRP can be changed by electric field. The study also showed that the activity index of HRP was increased by the certain strength of electric field. The activity index increase possibly was caused by the conformation variations of HRP. The result of this study has important meaning to explain the macroscopical electrostatic biological effects.

Poly(2-hydroxyethyl methacrylate) for Enzyme Immobilization: Impact on Activity and Stability of Horseradish Peroxidase

On the basis of their versatile structure and chemistry as well as tunable mechanical properties, polymer brushes are well-suited as supports for enzyme immobilization. However, a robust surface design is hindered by an inadequate understanding of the impact on activity from the coupling motif and enzyme distribution within the brush. Herein, horseradish peroxidase C (HRP C, 44 kDa), chosen as a model enzyme, was immobilized covalently through its lysine residues on a N-hydroxysuccinimidyl carbonate-activated poly(2-hydroxyethyl methacrylate) (PHEMA) brush grafted chemically onto a flat impenetrable surface. Up to a monolayer coverage of HRP C is achieved, where most of the HRP C resides at or near the brush-air interface. Molecular modeling shows that lysines 232 and 241 are the most probable binding sites, leading to an orientation of the immobilized HRP C that does not block the active pocket of the enzyme. Michaelis-Menten kinetics of the immobilized HRP C indicated little change in the K(m) (Michaelis constant) but a large decrease in the V(max) (maximum substrate conversion rate) and a correspondingly large decrease in the k(cat) (overall catalytic rate). This indicates a loss in the percentage of active enzymes. Given the relatively ideal geometry of the HRPC-PHEMA brush, the loss of activity is most likely due to structural changes in the enzyme arising from either secondary constraints imposed by the connectivity of the N-hydroxysuccinimidyl carbonate linking moiety or nonspecific interactions between HRP C and DSC-PHEMA. Therefore, a general enzyme-brush coupling motif must optimize reactive group density to balance binding with neutrality of surroundings.

Influence of Water Activity and System Mobility on Peroxidase Activity in Maltodextrin Solutions

Maltodextrins influenced the enzymatic activity in aqueous solutions by affecting the water activity (aw) and mobility as described by viscosity and T’g. In diluted solutions, viscosity being equal, (1) maltodextrin with dextrose equivalents (DE) of 33 was more effective than glucose in limiting horseradish peroxidase (HRP) activity; (2) an increase in the maltodextrin chain length from DE 33 to DE 8.7 did not further limited enzymatic activity; (3) the maltodextrin with the highest chain length (DE 2.5) determined the highest enzyme inhibition. In general, the increase of molecular weight negatively affected the HRP activity by increasing the viscosity and T’g (decreasing molecular mobility) but it positively affected the aw and, in some cases, this compensated the HRP activity inhibition. In concentrated solutions (apparent viscosity ≈ 40 mPa s) the HRP activity decreased with the increase of the maltodextrin molecular weight, and it showed a dependence on T’g which could be described by a William, Landel and Ferry (WLF)-type equation. On the contrary, in the solution added with the maltodextrin with the highest chain length (DE 2.5) the HRP activity was much higher than that predicted by the WLF-type equation. The maltodextrin with DE 2.5 contains intact starch fragments and in water forms a suspension. In such a discontinuous system, the viscosity in the vicinity of the protein is lower than the bulk viscosity, and thus, the enzyme activity is higher than expected. Moreover, since T’g is a property of the soluble phase, it does not explain the mobility in discontinuous systems.

Fluorescence Decrease of Conjugated Polymers by the Catalytic Activity of Horseradish Peroxidase and Its Application in Phenolic Compounds Detection

We report the fluorescence decrease of the water-soluble π-π-conjugated polymer poly(2-methoxy-5-propyloxy sulfonate phenylene vinylene, MPS-PPV) by the catalytic activity of horseradish peroxidase in the presence of H(2)O(2). MPS-PPV acts as a donor substrate in the catalytic cycle of horseradish peroxidase where the electron-deficient enzymatic intermediates compounds I and II can subtract electrons from the polymer leading to its fluorescence decrease. The addition of phenolic drug acetaminophen to the former solution favors the decrease of the polymer fluorescence, which indicates the peroxidase-catalyzed co-oxidation of MPS-PPV and acetaminophen. The encapsulation of horseradish peroxidase within polyacrylamide microgels allows the isolation of intermediates compound I and compound II from the polymer, leading to a fluorescence decrease that is only due to the product of biocatalytic acetaminophen oxidation. This system could be used to develop a new device for phenolic compounds detection.

Improved bacteria detection by coupling magneto-immunocapture and amperometry at flow-channel microband electrodes

This paper describes the first immunosensing system reported for the detection of bacteria combining immunomagnetic capture and amperometric detection in a one-step sandwich format, and in a microfluidic environment. Detection is based on the electrochemical monitoring of the activity of horseradish peroxidase (HRP), an enzyme label, through its catalysis of hydrogen peroxide (H 2O 2) in the presence of the mediator hydroquinone (HQ). The enzymatic reaction takes place in an incubation micro-chamber where the magnetic particles (MPs) are confined, upstream from the working electrode. The enzyme product is then pumped along a microchannel, where it is amperometrically detected by a set of microelectrodes. This design avoids direct contact of the biocomponents with the electrode, which lowers the risk of electrode fouling. The whole assay can be completed in 1 h. The experiments performed with Escherichia coli evidenced a linear response for concentrations ranging 10 2–10 8 cell ml −1, with a limit of detection of 55 cells ml −1 in PBS, without pre-enrichment steps. Furthermore, 100 cells ml −1 could be detected in milk, and with negligible interference by non-target bacteria such as Pseudomonas.

Influence of Nanoreactor Environment and Substrate Location on the Activity of Horseradish Peroxidase in Olive Oil Based Water-in-Oil Microemulsions

Oxidative enzymatic reactions using horseradish peroxidase (HRP) were carried out in water-in-oil (w/o) microemulsions composed of olive oil/lecithin/1-propanol/water, a model biomimetic system. The substrates used (gallic acid, octyl gallate and 2,2'-azino-bis[3-ethylbenzo-thiazoline-6-sulfonic acid] (ABTS)) have different hydrophobicities and possible locations in the microemulsion system. HRP reactivity with reference to substrate hydrophobicity and structural characteristics of the microemulsions is discussed. The nature of the enzyme microenvironments was examined using dynamic light scattering (DLS), differential scanning calorimetry (DSC) and diffusion NMR (DOSY) methodologies while the location of various enzymatic substrates in the microemulsion phase was assessed by solubility measurements and by taking pressure-area isotherms of mixed monolayers of the substrates with dipalmitoyl-phosphatidylcholine (DPPC), which is a major constituent of lecithin. In contrast to the bulk aqueous phase, in the severely restricted environment of the polar domains of the microemulsion HRP reacted faster with octyl gallate, a substrate that is solubilized at the lipid interfaces. HRP was deactivated in the olive oil microemulsions within a few hours, a phenomenon that has also been observed in other microemulsion systems.

Degradation of pentachlorophenol by a novel peroxidase-catalyzed process in the presence of reduced nicotinamide adenine dinucleotide

A novel horseradish peroxidase (HRP)-catalyzed H 2O 2 process in the presence of reduced nicotinamide adenine dinucleotide (NADH) was applied to remove aqueous pentachlorophenol (PCP). Parameters (pH, H 2O 2 concentration, HRP activity and NADH dosage) on PCP removal were investigated. It was found that initial 0.05 mM PCP was removed by 98% in HRP–NADH–H 2O 2 system at pH 5.0 and 30 °C for 1 h. Addition of O 2 in HRP–NADH–H 2O 2 system enhanced the removal rate of PCP due to promoting hydroxyl radicals ( OH) and superoxide anion radical ( O 2 - ) generation, which were confirmed by electron paramagnetic resonance (EPR)-spin trapping method. PCP removal efficiency decreased when O 2 - and H 2O 2 were scavenged by superoxide dismutase and catalase in HRP–NADH–O 2 system, indicating that OH/ O 2 - played a great role in the degradation of PCP. Gas chromatography–mass spectrometry (GC–MS) analysis revealed that octachlorinated dibenzodioxin (OCDD) in residual solution was reduced after treated by the HRP–NADH–O 2 process, resulting in lower toxicity of treated solution than conventional enzymatic process. Two enzymatic-catalysis pathways were proposed for PCP removal in HRP–NADH–H 2O 2/O 2 system: (i) OH/ O 2 - free radical oxidation (ii) conventional phenoxy polymerization.