Activity Of Horseradish Peroxidase Research Articles

Spectrofluorimetric determination of vitamin B1 using horseradish peroxidase as catalyst in the presence of hydrogen peroxide

In this work a new spectrofluorimetric method for the determination of vitamin B(1), based on the catalytic activity of horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2), has been developed. Non-fluorescent vitamin B(1) was easily converted through catalytic oxidation in alkaline medium into a fluorescent compound, even without exposure to light. The linear range for vitamin B(1) observed was 0.026-16.83 microg/mL (RSD = 1.75%). The correlation coefficient for the calibration curve and limit of detection were found to be 0.9964 and 0.015 microg/mL, respectively. The developed method is practical, simple, sensitive and relatively free from interference by coexisting substances and has been successfully applied for the determination of vitamin B(1) in pharmaceutical preparations.

The structure of horseradish peroxidase C characterized as a molten globule state after Ca 2+ depletion

The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca 2+ ions. Earlier studies suggested a critical role of one of the bound Ca 2+ ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca 2+-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca 2+-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca 2+, perturbs a H-bonding network connecting this Ca 2+ to the heme crevice that involves the distal water. The measured effects of Ca 2 + depletion can be interpreted as supporting a structural role for the distal Ca 2+ and for its enhanced significance in finetuning the protein structure to optimize enzyme activity.

Sensitive electrochemical detection of horseradish peroxidase at disposable screen-printed carbon electrode.

A rapid, simple and sensitive electrochemical assay of horseradish peroxidase (HRP) performed on disposable screen-printed carbon electrode was developed. HRP activities were monitored by square-wave voltammetric (SWV) measuring the electroactive enzymatic product in the presence of o-aminophenol and hydrogen peroxide substrate solution. SWV analysis demonstrated a greater sensitivity and shorter analysis time than the widely used amperometric and differential-pulsed voltammetric methods. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were optimized. Under optimized conditions, a linear response for HRP from 0.003 - 0.1 U/mL and a detection limit of 0.002 U/mL (1.25×10(-15) mol in 25 μL) were obtained with a good precision (RSD = 8%; n = 6). This rapid and sensitive HRP assay with microliters-assay volume could be readily integrated to portable devices and point-of-care (POC) diagnosis applications.

Chemiluminescence flow biosensor for glucose based on gold nanoparticle-enhanced activities of glucose oxidase and horseradish peroxidase

In this work, a novel chemiluminescence (CL) flow biosensor for glucose was proposed. Glucose oxidase (GOD), horseradish peroxidase (HRP) and gold nanoparticles were immobilized with sol–gel method on the inside surface of the CL flow cell. The CL detection involved enzymatic oxidation of glucose to d-gluconic acid and H 2O 2, and then the generated H 2O 2 oxidizing luminol to produce CL emission in the presence of HRP. It was found that gold nanoparticles could remarkably enhance the CL respond of the glucose biosensor. The enhanced effect was closely related to the sizes of gold colloids, and the smaller the size of gold colloids had the higher CL respond. The immobilization condition and the CL condition were studied in detail. The CL emission intensity was linear with glucose concentration in the range of 1.0 × 10 −5 mol L −1 to 1.0 × 10 −3 mol L −1, and the detection limit was 5 × 10 −6 mol L −1 (3 σ). The apparent Michaelis–Menten constant of GOD in gold nanoparticles/sol–gel matrix was evaluated to be 0.3 mmol L −1, which was smaller than that of GOD immobilized in sol–gel matrix without gold nanoparticles. The proposed biosensor exhibited short response time, easy operation, low cost and simple assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.

Activation and Inactivation of Horseradish Peroxidase by Cobalt Ions

The effect of cobalt ions (Co2+) on horseradish peroxidase (HRP) was studied in vitro by enzymatic activity assay, electronic absorption spectra, intrinsic fluorescence spectra and 8-anilo-1-naphthalenesulfonate(ANS)-binding fluorescence spectra. Co2+ at concentrations below 0.1 mM mildly increased the HRP activity, whereas higher concentrations of Co2+ significantly inactivated HRP in a time and concentration-dependent manner. Steady-state kinetic studies show that Co2+ was a noncompetitive inhibitor of o-dianisidine oxidation by HRP. The Ki value dropped as the incubation time increased. Furthermore, Co2+ was found to be an uncompetitive inhibitor of H2O2. These results suggested that Co2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectroscopic analysis showed that even for high Co2+ concentrations, the structure of HRP as a whole only changed slightly; however, there were significant conformational changes near or in the active site of HRP. Based on the above results, we suggest that Co2+ may bind with some amino acids near or in the active site of HRP and the conformational changes of HRP induced by such binding should be the main reason for activation and inactivation effect of Co2+. The potential binding sites of Co2+ were also proposed.

In vitro stimulatory effect of anti-apoptotic seminal vesicle protein 4 on purified peroxidase enzymes

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 mum native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme-electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.

Activity, stability, and unfolding of reconstituted horseradish peroxidase with modified heme

Heme-propionates of horseradish peroxidase (HRP) were esterified by p-nitrophenol, phenol and p-methylphenol to change its electron character and to increase its hydrophobicity. These synthetic hemes were inserted apo-HRP to give a novel HRP, respectively. Of the three reconstituted HRPs, reconstituted HRP with p-nitrophenol-modified heme derivative had a larger initial rate, affinity, catalytic efficiency and substrate-binding efficiency than native HRP in aqueous buffer and some solvents. The reconstituted HRPs showed higher thermostability and tolerance of DMF because of the increase of the hydrophobicity of the active site. Changing the electron character of the aromatic moieties linked at each terminal of the two heme-propionates can control activity and stability of HRP. The initial rate, affinity, catalytic efficiency and substrate-binding efficiency increased with the increases of electron-withdrawing efficiency of substituents at 4-position of the phenolic used to synthesize the heme derivatives, contrariwise, the stability decreased. The modifications resulted in the increase in the temperature ( T m) at the midpoint of thermal denaturation and the decreases in both enthalpy and entropy change at T m. The changes of catalytic properties and stabilities are related to the changes of the conformation of HRP. The modification changed the environment of heme and tryptophan, increased α-helix content of HRP. The present work demonstrates that enhancement of the hydrophobicity and the electron-withdrawing efficiency of heme improves the activity and stability of HRP.

Can amino acids protect horseradish peroxidase against its suicide-peroxide substrate?

Kinetics of horseradish peroxidase (HRP) in the oxidation reaction of ortho-methoxy phenol (AH) by hydrogen peroxide was studied taking into account inactivation of HRP during reaction by its suicide substrate, H 2O 2. A new simple method was introduced to protect HRP against suicidal inactivation effect of peroxide. Histidine, tyrosine and cysteine were added to protein solution and peroxidatic activity of HRP was assayed (25 °C, pH 7.0) under suicide inactivation condition, [H 2O 2] > 2 mM. Peroxidatic activity of HRP increased more than 1.5 times and HRP was protected against suicide inactivation, completely. A close match was observed for the proposed equations and experimental data. Surprisingly, inactivation rate constant ( k i) at 3.0 mM H 2O 2 was about zero in the presence of low-dose stabilizers of amino acids family ([amino acid]/HRP∼0.2–10.0) and stabilized HRP solutions were stable more than 45 days keeping 80% of the initial catalytic activity. A very interesting observation was that peroxide-inactivated HRP could be recovered into a more active one in the presence of amino acids. Such improvements in the catalytic activity, long-term stability and preventing suicide inactivation of HRP are of paramount importance in clinical assays, biosensors preparation and biotechnological applications of peroxidase (biotransformation, chemical synthesis and phenol removal process).

Direct Electrochemistry of Horseradish Peroxidase Immobilized in Calcium Carbonate Microsphere Doped with Phospholipids

AbstractProtein electrochemistry affords a direct method to study the biological electron transfer processes. However, supplying a biocompatible environment to maintain the native state of protein is all‐important and challengeable. Here, we chose vaterite, one of the crystalline polymorphs of calcium carbonate, with highly porous nature and large specific surface area, which was doped with phospholipids, as the matrix to immobilize horseradish peroxidase (HRP). The integrity of HRP was kept during the simple immobilization procedure. By virtue of this organic/inorganic complex matrix, the direct electrochemistry of HRP was realized, and the activity of HRP for catalyzing reduction of O2 and H2O2 was preserved.

Partition of horseradish peroxidase with maintained activity in aqueous biphasic system based on ionic liquid

Enzyme activity and partition behavior in aqueous biphasic systems (ABSs) consisting of ionic liquid (IL) and salt (IL-ABSs) were investigated to increase our understanding of IL-ABSs and shed light on their application potential as enzyme extraction system. With horseradish peroxidase (HRP) as the model enzyme, several effects of alkylimidazolium chloride–K 2HPO 4 ABSs on activity and partition behavior of enzyme were studied including alkyl chain length of ILs and concentrations of each component. High lyotropic ILs (1-butyl-3-methylimidazolium and 1-ethyl-3-methylimidazolium chloride) and adequate water content (>40%) were both essential for the activity maintenance of HRP in IL-ABS. 1-Butyl-3-methylimidazolium chloride ([C 4mim]Cl) was found to be an appropriate IL for phase forming and HRP activity retaining. After optimization of phase condition, about 80% HRP amount was distributed in the IL-rich upper phase, and greater than 90% enzyme activity was obtained. Moreover, compared with the commonly used polymer-based ABSs, this [C 4mim]Cl-ABS has a much lower viscosity, which is very beneficial to the experimental operation. Therefore, the tested IL-ABS could be considered as a potential enzyme extraction system.

Enhanced activity of horseradish peroxidase in Langmuir–Blodgett films of phospholipids

The immobilization of enzymes in nanostructured films has potential applications, e.g. in biosensing, for which the activity may not only be preserved, but also enhanced if optimized conditions are identified. Optimization is not straightforward because several requirements must be fulfilled, including a suitable matrix and film-forming technique. In this study, we show that horseradish peroxidase (HRP) has its activity enhanced when immobilized in Langmuir–Blodgett (LB) films, in conjunction with dipalmitoylphosphatidylglycerol (DPPG). Incorporation of HRP into a DPPG monolayer at the air–water interface was demonstrated with compression isotherms, and Polarization-Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS). From the PM-IRRAS data, we inferred that HRP was not denatured when adsorbed on a pre-formed, low pressure DPPG monolayer. A change in orientation was induced by the phospholipid matrix, with the amide C O and NH groups from HRP being oriented perpendicular to the surface, parallel to the DPPG acyl chains, i.e. the α-helix was inserted into the monolayer. The mixed DPPG-HRP monolayer could be transferred onto solid supports, to which HRP activity was ca. 23% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allowed HRP-containing LB films to be used in sensing peroxide.

High Catalytic Activities of Artificial Peroxidases Based on Supramolecular Hydrogels That Contain Heme Models

Composed of a supramolecular hydrogel and a heme model compound, a new type of artificial peroxidase shows high catalytic activity in organic media. The activity of this new type of artificial enzyme is significantly higher than that of the heme model compounds alone. Changes in the distal substituents above the coordinated-metal centers of the model compounds directly modulate catalytic activity. This supramolecular-hydrogel-based artificial enzyme is most active in toluene, reaching about 90% of the nascent activity of horseradish peroxidase. Moreover, this study confirms that the incorporation of the heme models into the nanofibers of gelators accounts for most of the enhancement of catalytic activity.

A third-generation hydrogen peroxide biosensor based on Horseradish Peroxidase (HRP) enzyme immobilized in a Nafion–Sonogel–Carbon composite

A third-generation biosensor based on HRP and a Sonogel–Carbon electrode has been fabricated with the aim of monitoring hydrogen peroxide in aqueous media via a direct electron transfer process. The redox activity of native HRP, typical of thin-layer electrochemistry, was observed. The charge coefficient transfer, α, and the heterogeneous electron transfer rate constant, k s, were calculated to be 0.51 ± 0.04 and 1.29 ± 0.04 s −1, respectively. Topographic study by atomic force microscopy (AFM) shows that the enzyme may have been introduced inside the ionic cluster of the Nafion. The immobilized HRP exhibited excellent electrocatalytical response to the reduction of H 2O 2 and preserved its native state after the immobilization stage. Several important experimental variables were optimized. The resulting biosensor showed a linear response to H 2O 2 over a concentration range from 4 to 100 μM, with a sensitivity of 12.8 nA/μM cm −2 and a detection limit of 1.6 μM, calculated as (3 S.D./sensitivity). The apparent Michaelis–Menten constant K m app was calculated to be 0.295 ± 0.020 mM. The biosensor showed high sensitivity as well as good stability and reproducibility. The performance of the biosensor was evaluated with respect to four possible interferences.

Attachment of horseradish peroxidase to polytetrafluorethylene (teflon) after plasma immersion ion implantation

The aim of this work was to investigate the potential of polytetrafluorethylene (PTFE) as a surface for biologically active protein attachment. A plasma immersion ion implantation (PIII) treatment was applied to PTFE to produce an activated surface for the functional attachment of the enzyme, horseradish peroxidase (HRP). Fourier transform infrared–attenuated total reflectance spectra show oxidation and carbonization of the surface layer as a function of ion fluence. The PIII treatment increases by threefold the amount of attached HRP and the activity of HRP on the modified surface is about seven times higher than that on an untreated PTFE surface. This result indicates that the PIII surface modification improves both the polymer’s protein binding capacity and its ability to retain the protein in a bioactive state.

Design and in vivo evaluation of solid-in-oil suspension for oral delivery of human growth hormone

Solid-in-oil (S/O) suspension containing human growth hormone (hGH), in which hGH was complexed with an edible surfactant, was designed and validated for oral administration of hGH. To optimize the formulation procedures, protein release behavior from the S/O suspensions under physiological conditions was first investigated with horseradish peroxidase (HRP) as a model protein. Evaluation of functional integrity of HRP released from the HRP–surfactant complex suggested that the solubilization process partly impaired the specific activity of HRP, however, the addition of trehalose in the formulation process regained up to about 50% of the biological activity. On the basis of the data collected with HRP, a surfactant–hGH complex prepared under optimized conditions was suspended in soybean oil to formulate S/O suspensions. After oral administration of the S/O suspension to male New Zealand rabbits, we detected hGH in the serum with 3.3% bioavailability, suggesting that hGH can be orally delivered to the systemic circulation by the present formulation.

An Amperometric Biosensor for trans‐Resveratrol Determination in Aqueous Solutions by Means of Carbon Paste Electrodes Modified with Peroxidase Basic Isoenzymes from Brassica Napus

AbstractThe catalytic properties of peroxidase basic isoenzymes (PBI's) from Brassica napus towards trans‐resveratrol (t‐Res) oxidation were demonstrated by the first time by conventional UV‐visible spectroscopic measurements. The enzymatic reaction rate was studied under different experimental conditions and the kinetics parameters were determined. An amperometric biosensor based on Brassica napus PBI's to determine t‐Res is also proposed by the first time. The method employs a dialysis membrane covered, PBI's entrapped and ferrocene (Fc)‐embedded carbon paste electrode (PBI's‐Fc‐CP) and is based on the fact that the decreased amount of H2O2 produced by the action of PBI's is proportional to the oxidised amount of t‐Res in the solution. Comparative amperometric experiments showed that, in spite of PBI's activity was much lower than commercial horseradish peroxidase (HRP) activity, t‐Res was a much better substrate for PBI's biosensors than those biosensors constructed by using HRP. The PBI's‐Fc‐CP biosensors showed a very good stability during at least twenty days. The reproducibility and the repeatability were 4.5% and 8.3%, respectively, showing a good biosensor performance. The calibration curve was linear in the t‐Res concentration (ct‐Res) range from 1×10−6 to 2.5×10−5 M, with a sensibility of (2.31±0.05)×106 nA M−1. The lowest ct‐Res value measured experimentally for a signal to noise ratio of 3 : 1 was 0.83 μM.

Sequential injection analysis as a tool for implementation of enzymatic assays in ionic liquids

An approach based on the use of water/1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4]) mixtures in a sequential injection analysis (SIA) system is presented. The rapid and robust procedure developed was used to evaluate horseradish peroxidase activity in [bmim][BF4] and is intended to be a generic tool for enzymatic assays in ionic liquids. The horseradish peroxidase activity tests were based on the implementation of the 4-aminoantipyrine (4AAP)/phenol test in the SIA system, using 1-naphtol as substrate. Small volumes (12 μL) of sample, reagents and enzyme were sequentially aspirated to the holding coil before being sent to the spectrophotometric detector ( λ = 510 nm), where a coloured product proportional to the enzyme activity was measured. The results were compared to those obtained when the assay was performed in water/methanol mixtures under the same conditions, to evaluate [bmim][BF4] as an alternative to conventional organic solvents. Comparative evaluation of the enzyme behaviour revealed that the enzyme activity increased significantly when the assay was performed in water/[bmim][BF4] mixtures. The SIA methodology exhibited good repeatability over the full concentration range (R.S.D. < 3.3%, n = 15) studied, produced approximately 1.7 mL of effluent and consumed approximately 36 μL of solutions prepared in water/[bmim][BF4] mixtures for each analytical cycle.

A Novel and Highly Sensitive Resonance Scattering Spectral Assay for Horseradish Peroxidase Using Cationic Surfactant

A novel and ultrasensitive resonance scattering (RS) spectral assay was proposed for detection of horseradish peroxidase (HRP) activity. It was based on that the HRP strongly catalyze H2O2 oxidation of excess I- to form I3-, the resulting I3- combined with four cationic surfactant (CS), including tetradecyl pyridinium bromide (TPB), cetyltrimethyl ammonium bromide (CTMA), cetylpyridinium chloride (CPCM) and tetradecyl dimethylbenzyl ammonium chloride (TDMBA) to produce association particles (TPB-I3)n, (CTMA-I3)m, (CPCM-I3)l and (TDMBA-I3)k, which exhibit a strongest resonance scattering peak at 478, 423, 538 and 491 nm, respectively. For the four systems of TPB, CPCM, CTMBA and TDMBA, the HRP activity determined was in the linear range of 0.004-5.6, 0.04-3.2, 0.04-8.0, 0.08-8.0 ng/mL, with a detection limit of 0.0034, 0.040, 0.033, 0.016 ng/mL, respectively. The TPB resonance scattering spectral assay was best and has been applied to the analysis of HRP in real samples, with satisfactory results.

Effects of sodium phosphate buffer on horseradish peroxidase thermal stability

Thermal stability of horseradish peroxidase (HRP) was studied by differential scanning calorimetry, tryptophan fluorescence, the heme absorption and enzymatic activity analysis while the concentrations of sodium phosphate buffer ranged from 2.5 to 50 mM at pH 7.0. The results showed that the denaturation temperature (Tm) values decreased and the intrinsic tryptophan fluorescence intensity of denatured HRP increased as sodium phosphate buffer concentration increased. Furthermore, the heme absorbance at 403 nm and enzymatic activity of HRP decreased with the increasing buffer concentrations. According to data obtained in this experiment, it can be concluded that sodium phosphate accelerated the denaturation process of HRP and reduced the thermal stability of HRP.

Effect of herbicide tralkoxydim and 2-acylcyclohexane-1,3-diones on peroxidase activity

Effect of 2-acylcyclohexane-1,3-dione derivatives (tralkoxydim and its diketone precursors) on peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (PDA), and the phenol-4-aminoantipyrine (4-AAP) couple has been studied. This effect varies from horseradish peroxidase (HRP) inactivation to activation in the reactions of peroxidation ofTMB, PDA, and, to a lesser extent, the phenol-4-AAP couple. The diketone-mediated HRP activation depends strongly on pH, presence of dimethylformamide, the structures of tralkoxydim and other diketones, and the substrate nature. The type of activation in the course of peroxidation with the presence of tralkoxydim can be noncompetitive (PDA and TMB) or mixed (TMB) depending on conditions. The maximal level of the HRP activation mediated by diketones depends on their structure. It can reach 4000% of the initial HRP-catalyzed peroxidation rate for TMB and ca. 1000% for PDA. A test system is proposed for quantitative tralkoxydim assay at millimolar concentration. It includes HRP and TMB as the substrate with spectrometrical monitoring of the TMB peroxidation product at 655 nm.