Activity Of Horseradish Peroxidase Research Articles

Effects of compressed fluids on the activity and structure of horseradish peroxidase

This work investigated the stability of commercial horseradish peroxidase in compressed carbon dioxide and propane. Effects of temperature, exposure time and pressure on the enzyme activity were evaluated. The results obtained indicated that the HRP treatment in CO 2 promotes a significant decrease in the enzyme activity, whereas HRP showed good stability in propane, indicating that this enzyme is more stable in propane than in CO 2. Far UV-circular dichroism (CD) analyses of HRP in solution indicated that treatment of the enzyme with propane did not induce changes in the secondary structure content of HRP. On the other hand, incubation with CO 2 led to significant loss of HRP secondary structure. The solid HRP did not present decrease of its activity after treatment with pressurized CO 2 or propane. The CD analysis of solid HRP treated with propane and CO 2 revealed that the enzyme once more present higher stability in propane, but the treatment in both solvents provoked loss in secondary structure.

Hyperhomocysteinemia Increases β-Amyloid by Enhancing Expression of γ-Secretase and Phosphorylation of Amyloid Precursor Protein in Rat Brain

Hyperhomocysteinemia and beta-amyloid (Abeta) overproduction are critical etiological and pathological factors in Alzheimer disease, respectively; however, the intrinsic link between them is still missing. Here, we found that Abeta levels increased and amyloid precursor protein (APP) levels simultaneously decreased in hyperhomocysteinemic rats after a 2-week induction by vena caudalis injection of homocysteine. Concurrently, both the mRNA and protein levels of presenilin-1, a component of gamma-secretase, were elevated, whereas the expression levels of beta-secretase and presenilin-2 were not altered. We also observed that levels of phosphorylated APP at threonine-668, a crucial site facilitating the amyloidogenic cleavage of APP, increased in rats with hyperhomocysteinemia, although the phosphorylation per se did not increase the binding capacity of pT668-APP to the secretases. The enhanced phosphorylation of APP in these rats was not relevant to either c-Jun N-terminal kinase or cyclin-dependent kinase-5. A prominent spatial memory deficit was detected in rats with hyperhomocysteinemia. Simultaneous supplementation of folate and vitamin-B12 attenuated the hyperhomocysteinemia-induced abnormal processing of APP and improved memory. Our data revealed that hyperhomocysteinemia could increase Abeta production through the enhanced expression of gamma-secretase and APP phosphorylation, causing memory deficits that could be rescued by folate and vitamin-B12 treatment in these rats. It is suggested that hyperhomocysteinemia may serve as an upstream factor for increased Abeta production as seen in patients with Alzheimer disease.

Acetylene plasma coated surfaces for covalent immobilization of proteins

A modified plasma enhanced chemical vapor method was used for acetylene plasma polymerization of biocompatible surfaces on a range of substrates. Smooth polymerized surfaces with excellent mechanical properties were achieved suitable for a wide range of biochemical and biomedical applications. Horseradish peroxidase activity analysis showed that the proteins immobilized on the plasma polymerized surfaces maintained their biological function for a much longer period of time compared to untreated surfaces. The plasma polymerized surfaces and the protein immobilization were also analyzed using quartz crystal microbalance with dissipation analysis, spectroscopic ellipsometry, X-ray photoelectron spectroscopy, and tensile strength analysis. The results indicate that the plasma polymerized surfaces provide covalent bonding sites and immobilize a dense monolayer of proteins after incubation in protein containing solution.

Electrochemical detection of glyphosate herbicide using horseradish peroxidase immobilized on sulfonated polymer matrix

This paper describes the use of horseradish peroxidase (HRP) based biosensor for novel detection of glyphosate herbicide. The biosensor was prepared by electrochemically depositing poly(2,5-dimethoxyaniline) (PDMA) doped with poly(4-styrenesulfonic acid) (PSS) onto the surface of a gold electrode followed by electrostatic attachment of the enzyme HRP onto the PDMA-PSS composite film. Fourier transform infrared (FTIR) and UV–Vis spectrometry inferred that HRP was not denatured during its immobilization on PDMA-PSS composite film. The biosensing principle was based on the determination of the cathodic responses of the immobilized HRP to H 2O 2, before and after incubation in glyphosate standard solutions. Glyphosate inhibited the activity of HRP causing a decrease in its response to H 2O 2. The determination of glyphosate was achieved in the range of 0.25–14.0 µg L − 1 with a detection limit of 1.70 μg L − 1 . The apparent Michaelis–Menten constant (calculated for the HRP/PDMA-PSS biosensor in the presence and absence of glyphosate was found to be 7.73 µM and 7.95 µM respectively.

Enzyme activity of horseradish peroxidase immobilized in chitosan matrices in alternated layers

The successful immobilization of enzymes such as horseradish peroxidase (HRP) in solid films is essential for applications in sensors and for fundamental studies aimed at identifying possible biotechnological devices. In this study we show that HRP can be immobilized in alternated layers with chitosan as the template material. The activity of HRP in HRP/chitosan films was preserved for several weeks, and could be detected optically upon monitoring the reaction with pyrogallol. The morphology of the film displayed stripes that disappeared after reaction with pyrogallol. Though the activity in the HRP/chitosan film was lower than in a homogeneous solution or in an LB film investigated earlier, the response was linear for a considerable period of time, which may be advantageous for sensing hydrogen peroxide.

A microfluidic electrochemical device for high sensitivity biosensing: Detection of nanomolar hydrogen peroxide

We report herein a simple device for rapid biosensing consisting of a single microfluidic channel made from poly(dimethylsiloxane) (PDMS) coupled to an injector, and incorporating a biocatalytic sensing electrode, reference and counter electrodes. The sensing electrode was a gold wire coated with 5 nm glutathione-decorated gold nanoparticles (AuNPs). Sensitive detection of H 2O 2 based on direct bioelectrocatalysis by horseradish peroxidase (HRP) was used for evaluation. HRP was covalently linked the glutathione–AuNPs. This electrode presented quasi-reversible cyclic voltammetry peaks at −0.01 V vs. Ag/AgCl at pH 6.5 for the HRP heme Fe III/Fe II couple. Direct electrochemical activity of HRP was used to detect H 2O 2 at high sensitivity with a detection limit of 5 nM in an unmediated system.

Characterization of Multienzyme-Antibody-Carbon Nanotube Bioconjugates for Immunosensors

Characterization studies of a multi-enzyme-antibody-carbon nanotube bioconjugate designed for the amplification of electrochemical immunosensing are described. Secondary antibodies for prostate specific antigen (PSA) were covalently linked to highly carboxylated multi-walled carbon nanotube (CNT) along with multiple horseradish peroxidase (HRP) enzyme labels. These bioconjugates provide ultra-sensitive amperometric detection of PSA on a single-wall carbon nanotube forest sandwich immunosensor platform. A single layer of HRP on the surface of the CNT was suggested by images from atomic force microscopy (AFM) and transmission electron microscopy (TEM). HRP on the bioconjugate surface was visualized by confocal microscopy using in-situ HRP-catalyzed polymerization yielding a fluorescent product, and HRP activity was estimated in a conventional assay. Binding of quantum-dot labeled PSA to antibodies on the bioconjugate was used for visualization by TEM. Combining TEM and enzyme activity results gave estimates of approximately 82 HRPs and 30 +/- 15 secondary antibodies per 100 nm of antibody-HRP-CNT bioconjugate.

Enzymatic activity of surface-immobilized horseradish peroxidase confined to micrometer- to nanometer-scale structures in nanocapillary array membranes

Horseradish peroxidase (HRP) was immobilized on the planar surfaces and inside the cylindrical nanopores of nanocapillary array membranes (NCAMs) to study how the enzyme-catalyzed oxidation of a fluorigenic substrate, Amplex Red (AR), to fluorescent resorufin by hydrogen peroxide is influenced by confinement. Because AR was also found to be converted to resorufin photolytically at high laser fluences, a modified laser-induced fluorescence protocol was developed to characterize the enzyme-catalyzed reaction in the absence of interference from the photolytic reaction. Surface-immobilized HRP was studied in two environments: bound to the surface of a microfluidic channel, and bound to the interior of cylindrical nanopores in NCAMs connecting crossed microfluidic channels. HRP was immobilized through reaction of solvent-accessible primary amines with the epoxy group of the methyl methacrylate-glycidyl methacrylate copolymer synthesized in either planar or annular geometries to construct the test structures for enzymatic activity. HRP immobilized on planar surfaces shows high activity (approximately 10 microM min(-1)) meaning that the copolymer membrane exhibits good potential for immobilizing the enzyme, especially since active structures are obtained in a one-step reaction. HRP was also immobilized inside nanopores via physisorption. Enzymatic reactions inside the nanopores were characterized and compared to finite element simulations of a modified Eley-Rideal mechanism to bracket the value of the overall rate constant for the confined enzyme. Reaction velocities were estimated to be approximately 10-fold higher in the nanopores than for the same enzyme bound to a planar microfluidic surface.

Fluorometric Determination of Quinolinic Acid Using the Catalytic Activity of Horseradish Peroxidase

The conversion of quinolinic acid (QA) into a fluorescent compound by the catalytic activity of horseradish peroxidase (HRP) was investigated in the presence of hydrogen peroxide without exposure to light. Nonfluorescent quinolinic acid was converted into a fluorescent compound with maximum excitation and emission wavelengths at 328 and 377 nm, respectively. This fluorescent derivatization reaction with HRP in the presence of hydrogen peroxide was adopted for the determination of trace amounts of QA. The calibration curve obtained was linear from 0.1 to 5.0 nmol of QA in a 1.0 ml sample solution. The detection limit was 0.04 nmol/ml. The relative standard deviation at 3.0 nmol of QA was 3.58% (n = 8). This method was applied to the determination of QA spiked in control serum I and II. The recovery rates of QA were greater than 98%, and satisfactory results were obtained for both control serum I and II.

Photocatalytic activity of colloidal CdS nanoparticles with different capping ligands

We report on the synthesis and characterization of cadmiumsulfide quantum dots (CdS-QDs) with three different types of capping ligands, mercaptoacetic acid (CdS-MAA), cysteamine, and 2-mercaptoethanesulfonic acid (CdS-CM), or tiopronin (CdS-TPN). All three types of QDs generate reactive oxygen species (ROS), in particular, superoxide- and hydroxyl radicals, upon irradiation with UV light. The ROS were analyzed using electron paramagnetic resonance (EPR) spectroscopy. Moreover, the hydroxyl radical generation from the three different QDs was monitored using a terephthalate-based fluorimetric assay. The three QDs were then used as photocatalyst to trigger the peroxidase activity of horseradish peroxidase.

Biocatalysis in Water-in-Ionic Liquid Microemulsions: A Case Study with Horseradish Peroxidase

In this article we report the first results on the enzymatic activity of horseradish peroxidase (HRP) microencapsulated in water-in-ionic liquid (w/IL) microemulsions using pyrogallol as the substrate. Toward this goal, the system used in this study was composed of anionic surfactant AOT (sodium bis(2-ethyl-1-hexyl)sulfosuccinate)/hydrophobic IL [C(8)mim][Tf(2)N] (1-octyl-3-methyl imidazolium bis(trifluoromethylsulfonyl)amide)/water/1-hexanol. In this system, the catalytic activity of HRP was measured as a function of substrate concentrations, W(0) (molar ratio of water to surfactant), pH, and 1-hexanol content. The curve of the activity-W(0) profile was found to be hyperbolic for the new microemulsion. The apparent Michaelis-Menten kinetic parameters (k(cat) and K(m)) were estimated and compared to those obtained from a conventional microemulsion. Apparently, it was found that HRP-catalyzed oxidation of pyrogallol by hydrogen peroxide in IL microemulsuions is much more effective than in a conventional AOT/water/isooctane microemulsion. The stability of HRP solubilized in the newly developed w/IL microemulsions was examined, and it was found that HRP retained almost 70% of its initial activity after incubation at 28 degrees C for 30 h.

Single-crystal CeO2 nanocubes used for the direct electron transfer and electrocatalysis of horseradish peroxidase

A new horseradish peroxidase (HRP) third-generation electrochemical biosensor based on ceria nanocubes (CeO2-NCs) and chitosan (Chit) was developed. The single-crystalline, uniform and size-controlled CeO2-NCs have been synthesized by hydrothermal method. HRP was immobilized in CeO2-NCs and Chit film on the glass carbon electrode (HRP/CeO2/Chit/GCE). Compared with HRP-chitosan modified electrode (HRP/Chit/GCE), HRP/CeO2/Chit/GCE exhibited a pair of more obvious redox peaks at −0.348V (versus Ag/AgCl). Experimental results indicate CeO2-NCs greatly promoted the electron transfer between HRP and GCE. The immobilized HRP exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide (H2O2). The resulting biosensor showed a linear range of 1–150μM and a detection limit of 0.26μM estimated at a signal-to-noise ratio of 3. Stability and reproducibility of the biosensor were also studied. The biological activity of HRP immobilizing in the composite film was characterized by UV–vis and FTIR spectra.

Amperometric hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase on core–shell organosilica@chitosan nanospheres and multiwall carbon nanotubes composite

The application of the composites of multiwall carbon nanotubes (MWNTs) and core–shell organosilica@chitosan crosslinked nanospheres as an immobilization matrix for the construction of an amperometric hydrogen peroxide (H 2O 2) biosensor was described. MWNTs and positively charged organosilica@chitosan nanospheres were dispersed in acetic acid solution (0.6 wt%) to achieve organosilica@chitosan/MWNTs composites, which were cast onto a glass carbon electrode (GCE) surface directly. And then, horseradish peroxidase (HRP), as a model enzyme, was immobilized onto it through electrostatic interaction between oppositely charged organosilica@chitosan nanospheres and HRP. The direct electron transfer of HRP was achieved at HRP/organosilica@chitosan/MWNTs/GCE, which exhibited excellent electrocatalytic activity for the reduction of H 2O 2. The catalysis currents increased linearly to H 2O 2 concentration in a wide range of 7.0 × 10 −7 to 2.8 × 10 −3 M, with a sensitivity of 49.8 μA mM −1 cm −2 and with a detection limit of 2.5 × 10 −7 M at 3 σ. A Michaelies–Menten constant K M a p p value was estimated to be 0.32 mM, indicating a high-catalytic activity of HRP. Moreover, the proposed biosensor displayed a rapid response to H 2O 2 and possessed good stability and reproducibility. When used to detect H 2O 2 concentration in disinfector samples and sterilized milks, respectively, it showed satisfactory results.

Effects of different valences of cerium ion on conformation of Horseradish Peroxidase

Our previous studies demonstrated that Ce 4+ could induce reactive oxygen species (ROS) burst as a signal to promote pacilitaxel biosynthesis in suspension cultured Taxus cuspidate cells. To further understand the mechanism of cerium ions inducing ROS burst, circular dichroism (CD), synchronous fluorescence, and electron paramagnetic resonance (EPR) were used to detect them inducing conformational change of horseradish peroxidase (HRP). Horseradish peroxidase activity was reduced by 78% by 0.1 mmol/L Ce 4+, whereas it was only reduced by 28% by 0.1 mmol/L Ce 3+. Circular dichroism spectra showed that the percentage of transition from helical content and other structure to β strands and β turns was 23.1 when induced by Ce 4+, whereas it was only 13.2 when induced by Ce 3+. In synchronous fluorescence spectra, Ce 4+ led to red shift and intensity-elevation of tryptophan fluorescence emission maximum, whereas in the case of Ce 3+, the results were a contrast to the above. Furthermore, g factor ( g x and g y ) in electron paramagnetic resonance (EPR) induced by Ce 4+ and Ce 3+ was significantly different. These results indicated that the different valence of cerium ion induced various conformations of HRP, and Ce 4+ was more effective than Ce 3+. This suggested that Ce 4+ affected the burst of ROS through changing the conformation of oxidoreductase.

Enzymatic synthesis of water‐soluble conducting poly(3,4‐ethylenedioxythiophene): A simple enzyme immobilization strategy for recycling and reusing

The field of intrinsically conducting polymers has attracted high attention due to their interesting electrical and optical properties. Since their discovery in 1977, they have been investigated for many technological applications, such as organic lightweight batteries, microelectronics, optical displays, antistatic coatings, sensors, direct metallization, all-polymer actuators, and electromagnetic shielding. These highly promising materials have traditionally been synthesized by monomer oxidation in the presence of a strong oxidant and, once synthesized, they usually show high insolubility and intractability. The use of enzymes as biocatalysts for the synthesis of conducting polymers has been studied in last years as a ‘‘green synthesis process’’ alternative. Enzymes can offer environmentally benign reaction conditions, a high yield of polymerization, and higher control in regioregularity and stereochemistry, consequently resulting in soluble and processable conducting polymers. In most cases, the research was focused on the synthesis of polyaniline (PANI). According to this biocatalytic approach, the aniline is coupled together by horseradish peroxidase (HRP) or another peroxidase catalyzed oxidation in the presence of an anionic polyelectrolyte or a micellar template leading to a PANI aqueous dispersion. Moreover, in general, the expensive enzymes must be recovered and reused after the reaction for practical applications. This is the main reason behind the well established strategy of immobilization of enzymes into solid supports which was applied to the HRP enzyme. Recently, our group reported the first enzymatic polymerization of 3,4-ethylenedioxythiophene (EDOT), demonstrating that this green synthesis process can be applied to other technologically interesting polymers besides PANI. In this work, a pH of 2 and a Temperature of 4 C were established as optimum conditions for the adequate synthesis of PEDOT. And even if some previous researches reported an abrupt decrease of the enzymatic activity in acidic media (at pH 1⁄4 4, the HRP activity is around 0 after 1 h), in our case, the reaction was observed to proceed uninterruptedly throughout 16 h. Then, we proved that the enhanced enzyme activity was due to the presence of an excess of EDOT in the media (monomer droplets), where the HRP was more preferentially localized. Monomer droplets would act, consequently, as (a) enzyme protectors against deactivation as well as (b) a monomer feed to keep a constant EDOT concentration in the reaction media. In another previous contribution by the authors a biphasic catalytic system was employed as a way to recover and reuse the enzyme several times after aniline polymerization. In this case, the enzyme was encapsulated inside an ionic liquid while the aniline monomer, the H2O2 oxidant, and the template stayed in aqueous Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 47, 306–309 (2009)

Direct electrochemistry and enhanced electrocatalysis of horseradish peroxidase based on flowerlike ZnO–gold nanoparticle–Nafion nanocomposite

We developed a new flowerlike ZnO–gold nanoparticles (GNPs)–Nafion nanocomposite which can promote the direct electron transfer of horseradish peroxidase (HRP) immobilized in the film effectively. The cyclic voltammetric results show that HRP entrapped in the composite could undergo a direct quasi-reversible electrochemical reaction with a formal potential ( E 0′) of −0.32 V (versus Ag/AgCl) in phosphate buffer solution (PBS). The flowerlike ZnO and GNPs show synergistic effect and the ZnO–GNPs–Nafion–HRP modified GC electrode gives an enhanced electrocatalytic activity towards the reduction of hydrogen peroxide (H 2O 2). The calculated apparent Michaelis–Menten constant ( K m app ) is 1.76 mM, which is much lower than that reported, indicating a high catalytic activity of HRP. The catalysis currents increase linearly to the H 2O 2 concentration in a wide range of 1.5 × 10 −5 to 1.1 × 10 −3 M, with a correlation coefficient of 0.998. The detection limit is 9.0 × 10 −6 M (at the ratio of signal to noise, S/N = 3), demonstrating that the formed film provides a favorable microenvironment for the enzyme to retain its activity. Moreover, the modified electrode displays rapid response to H 2O 2 and possesses good stability and reproducibility.

Acetylene plasma polymerized surfaces for covalent immobilization of dense bioactive protein monolayers

Smooth polymerized surfaces, suitable for biochemical and biomedical applications, were deposited using a modified plasma enhanced chemical vapour deposition method with acetylene as a reaction precursor. Horseradish peroxidase (HRP) activity assays showed that the protein immobilized on the plasma polymerized surfaces maintained its biological function for a much longer period of time compared to that on uncoated surfaces. The kinetics of HRP attachment to the plasma polymerized surfaces were analyzed using quartz crystal microbalance with dissipation analysis. Spectroscopic ellipsometry and attenuated total reflection Fourier transform infrared spectroscopy were used to determine the thickness and the quantity of the attached protein. The results showed that the plasma polymerized surfaces provided a high density of attachment sites to covalently immobilize a dense monolayer of proteins.

Nitric Oxide and Nitroxides Can Act as Efficient Scavengers of Protein-Derived Free Radicals

Nitric oxide ((*)NO) may act as either a pro-oxidant or an antioxidant in biological systems. Although (*)NO and nitroxide radicals react slowly with most molecules, they react at near diffusion-controlled rates with other radicals and may therefore be efficient protective agents. This study assessed the ability of (*)NO and nitroxides to intercept specific protein-derived radicals and compared the efficacy of these species. Three protein radical systems were investigated as follows: BSA-derived radicals generated via radical transfer from H(2)O(2)-activated horseradish peroxidase, radicals formed on myoglobin via reaction with H(2)O(2), and carbon-centered radicals formed from amino acid hydroperoxides on exposure to Fe(2+)-EDTA. In each case, radicals were generated in the absence or presence of (*)NO or nitroxides of different size and charge. Concentration-dependent loss of the protein radicals was detected by electron paramagnetic resonance with both (*)NO and nitroxides and time-dependent consumption of (*)NO using an (*)NO electrode. The protein oxidation product dityrosine was significantly reduced by (*)NO and nitroxides, and 3,4-dihydroxyphenylalanine levels were reduced by nitroxides but not (*)NO. Overall, these studies demonstrate that (*)NO and nitroxides are efficient near-stoichiometric scavengers of protein radicals and, hence, are potential protective agents against protein oxidation reactions and resulting damage. These reactions show little dependence on nitroxide structure or charge.

Improved direct electron transfer and electrocatalytic activity of horseradish peroxidase immobilized on gemini surfactant–polyvinyl alcohol composite film

The voltammetric and electrocatalytic behavior of horseradish peroxidase (HRP) immobilized on a cationic gemini surfactant (i.e. C 12H 25N(CH 3) 2–C 12H 24–N(CH 3) 2C 12H 25Br 2, C 12–C 12–C 12)–polyvinyl alcohol (PVA) composite film-coated glassy carbon electrode (GCE) has been studied. It is found that on the novel composite film HRP presents excellent electroactivity and can exhibit a pair of well-defined voltammetric peaks in 0.10 M pH 7.0 phosphate buffer solution (PBS). The immobilized HRP also presents good bioelectrocatalytic activity, and it can catalyze the reduction of oxygen (O 2), hydrogen peroxide (H 2O 2), nitrite ion (NO 2 −) and trichloroacetic acid (TCA). For H 2O 2 the catalytic current is linear to its concentration in the range of 0.195–97.5 μM, and the detection limit is down to 6.5 × 10 −8 M. The response shows Michaelis–Menten feature and the apparent Michaelis–Menten constant is estimated to be 110.5 μM. Similarly, the electrode can sense NO 2 − and TCA. In addition, it is observed that the spacer group of gemini surfactant affects the electroactivity of HRP significantly. A spacer group with higher flexibility and hydrophility is favorable to the electron transfer of HRP. UV–vis spectrum indicates that the structure of HRP in the PVA–C 12–C 12–C 12 film is similar to that of native HRP. Thus the C 12–C 12–C 12–PVA composite possesses good biocompatibility and has promising application in fabricating biosensor and bioelectronics.

Magnetically Enhanced Dielectrophoretic Assembly of Horseradish Peroxidase Molecules: Chaining and Molecular Monolayers

Synergy in assembly: The combination of a magnetostatic field with dielectrophoresis can assemble biological molecules—something not possible when these techniques are used in isolation. The AFM image shows horseradish peroxidase (HRP) monolayers. Electromagnetic treatment does not affect the molecular structure, as indicated by the uninhibited catalytic activity of HRP after treatment.