Activity Of Horseradish Peroxidase Research Articles

Effect of pulsed light on activity and structural changes of horseradish peroxidase

The objective of this research was to investigate the effects of pulsed light on the activity and structure of horseradish peroxidase in buffer solution. Enzyme residual activities were measured. Surface topography, secondary, and tertiary structures of horseradish peroxidase were determined using atomic force microscopy (AFM), Raman spectroscopy, and fluorescence spectroscopy, respectively. Results showed that a complete inactivation of horseradish peroxidase was achieved by application of 10 pulses of pulsed light treatment at an intensity of 500J/pulse. The AFM analysis revealed that the aggregation of enzyme protein increased and surface roughness decreased with the increase in the treatment time. Fluorescence and Raman spectroscopy analysis exhibited that pulsed light destroyed the tertiary and secondary protein structures. The β-sheet composition was decreased while β-turn and random coils were increased. Pulsed light could effectively inactivate horseradish peroxidase by destroying the secondary and tertiary structures of protein in the active center of the enzyme.

PH-sensitive pHluorins as a molecular sensor for in situ monitoring of enzyme-catalyzed prodrug activation.

This work examines the feasibility of using a pH-sensitive fluorescent protein as a molecular reporter for enzyme-catalyzed prodrug activation reaction. Specifically, a ratiometric pHluorins was examined for detection of the activity of horseradish peroxidase (HRP) for the activation of indole-3-acetic acid. The pHluorins and HRP were conjugated chemically, forming a biocatalyst with a self-reporting function. Results showed that the characteristic fluorescence intensity ratio of the conjugate shifted from 1.47 to 1.40 corresponding to the progress of the prodrug activation reaction. The effectiveness of applying the conjugate for inhibition of the growth of Bcap-37 cells was also demonstrated simultaneously with reaction monitoring. The results reveal a very promising approach to realizing in situ monitoring of enzyme activities based on pH shifting for enzyme-based prodrug therapy applications.

A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli

Horseradish peroxidase (HRP) is used in various biotechnological and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither production from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titres of 10mg per litre cultivation broth. Thus, the traditional way of production of HRP from plant still prevails. In this study, we revisited the recombinant production of HRP in E. coli and investigated and compared both strategies, (a) the production of HRP as inclusion bodies (IBs) and subsequent refolding and (b) the production of active HRP in the periplasm. In fact, we were able to produce HRP in E. coli either way. We obtained a refolding yield of 10% from IBs giving a final titre of 100mgL−1 cultivation broth, and were able to produce 48mg active HRP per litre cultivation broth in the periplasm. In terms of biochemical properties, soluble HRP showed a highly reduced catalytic activity and stability which probably results from the fusion partner DsbA used in this study. Refolded HRP showed similar substrate affinity, an 11-fold reduced catalytic efficiency and 2-fold reduced thermal stability compared to plant HRP. In conclusion, we developed a toolbox for HRP engineering and production. We propose to engineer HRP by directed evolution or semi-rational protein design, express HRP in the periplasm of E. coli allowing straight forward screening for improved variants, and finally produce these variants as IB in high amounts, which are then refolded.

Colorimetric biosensor for the assay of paraoxon in environmental water samples based on the iodine-starch color reaction

In this work, a new colorimetric biosensor for the assay of paraoxon was developed via the conventional iodine-starch color reaction and multi-enzyme cascade catalytic reactions. In the presence of acetylcholine chloride, acetylcholinesterase (AChE) and choline oxidase (ChO) catalyzed the formation of H2O2, which then activated horseradish peroxidase (HRP) to catalyze the oxidation of KI to produce an iodine-starch color reaction. Upon exposure to paraoxon, the catalytic activity of AChE was inhibited and less H2O2 generated, resulting in a decrease in the production of I2 and a drop in the intensity of solution color. This colorimetric biosensor showed high sensitivity for the assay of paraoxon with a limit of detection 4.7 ppb and was applied for the assay of paraoxon in spiked real samples. By employing the conventional iodine-starch color reaction, this biosensor has the potential of on-site assay of OPs residues in environmental samples.

Amorphophallus paeoniifolius corm: A potential source of peroxidase for wide applications

ABSTRACTPeroxidases have wide applications in different areas including chemical synthesis, medicine, food industry, bioremediation of wastewater, biosensing, and biotechnology. Amorphophallus paeoniifolius (elephant foot yam) is an attractive source of enzymes; however, no effort has been made to assess peroxidase enzyme from this source. Therefore, the objectives of this study were to evaluate and compare peroxidases from corms of elephant foot yam and roots of Dacus carota (carrot) and Aramoracia rusticana (horseradish). Elephant foot yam corm peroxidase (ECP) demonstrated 4.5 times higher specific activity compared with carrot root peroxidase (CRP). ECP showed retention of high activity over a broad pH range and had higher temperature optima and thermal stability compared with CRP and horseradish peroxidase (HRP). The calculated KM values of ECP, CRP, and HRP for the substrates guaiacol and hydrogen peroxide were 4.5 mM and 307.8 µM, 4.6 mM and 55.5 µM, and 3.5 mM and 413.0 µM, respectively. Peroxidases are used for the bioremediation of wastewater contaminated with hazardous aromatic compounds. Heavy metals such as cadmium (Cd2+), lead (Pb2+), and toxic compounds such as sodium azide (NaN3) are often present in wastewater along with aromatic compounds. Results showed activation of ECP and inhibition of HRP at 250 and 500 µM concentrations of Cd2+ and Pb2+. Treatment with 1 mM NaN3 led to 8.4%, 55.5%, and 11.0% inhibition in the activities of ECP, CRP, and HRP, respectively. Overall, our results suggest that elephant foot yam can be used as a rich and convenient source of peroxidase for various applications including wastewater remediation.

Horseradish peroxidase immobilized on copper surfaces and applications in selective electrocatalysis of p-dihydroxybenzene

Horseradish Peroxidase (HRP) was immobilized on copper surfaces with the linker of L-Cysteine (L-Cys) self-assembled films to form Cu/L-Cys/HRP electrodes. The activity of HRP can be preserved by the Cu/L-Cys self-assembled films. The Cu/L-Cys/HRP electrodes can be used for the selective electrocatalytic oxidase of p-dihydroxybenzen in absent of H2O2. The optimum pH for electrocatalyzing p-dihydroxybenzen was 5.5 or 7.0, which corresponds to the isoelectric points of L-Cys and HRP, respectively. X-ray photoelectron spectroscopy (XPS) provided the evidence that L-Cys linked with Cu surface by the Cu S bond. Fourier transform infrared spectroscopy (FTIR) analyses indicated that aromatic plane of p-dihydroxybenzen was connected parallel to porphyrin ring of heme in HRP. Quantum chemical calculation of density functional theory (DFT) revealed that symmetry of molecular structure and minimum space steric hindrance for p-dihydroxybenzen were benefit to combination with HRP. Moreover, the lowest energy of LUMO and most negative charges of oxygen atom on hydroxyl group of p-dihydroxybenzen were advantage to lose the hydrogen atom of hydroxyl group to be oxided.

High-Throughput Screening for Readthrough Modulators of CFTR PTC Mutations.

Cystic fibrosis (CF) is a hereditary disease caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). A large number of nearly 2000 reported mutations, including the premature termination codon (PTC) mutations, urgently require new and personalized medicines. We have developed cell-based assays for readthrough modulators of CFTR PTC mutations (or nonsense mutation suppressors), based on the trafficking and surface expression of CFTR. Approximately 85,000 compounds have been screened for two PTC mutations (Y122X and W1282X). The hit rates at the threshold of 50% greater than vehicle response are 2% and 1.4% for CFTR Y122X and CFTR W1282X, respectively. The overlap of the two hit sets at this stringent hit threshold is relatively small. Only ~28% of the hits from the W1282X screen were also hits in the Y122X screen. The overlap increases to ~50% if compounds are included that in the second screen achieve only a less stringent hit criterion, that is, horseradish peroxidase (HRP) activity greater than three standard deviations above the mean of the vehicle. Our data suggest that personalization may not need to address individual genotypes, but that patients with different CFTR PTC mutations could benefit from the same medicines.

Hooking horseradish peroxidase by using the affinity Langmuir-Blodgett technique for an oriented immobilization

A novel method of oriented immobilization was presented: affinity Langmuir-Blodgett (LB) technique. Firstly, a long carbon chain was bond to a ligand of Horseradish Peroxidase (HRP). The ligand derivative appears surface activity with the hydrophobic carbon chain oriented to air and the hydrophilic ligand faced to water. Then, this derivative was put onto the water/air surface to assemble a LB film and formed the affinity interaction with the active site of HRP. After that, the affinity LB film with the enzyme was transferred onto the support to obtain the oriented immobilized HRP. The specific activity of HRP immobilized by affinity LB (182.1±14U/mg) was higher than that by adsorption (40.5±5U/mg). HRP immobilized by affinity LB could maintain a more native conformation, compared to that by adsorption. This method could be effectively used to immobilize protein with orientation and show widely promising applications in many fields including biosensor and bioreactor.

Spectrometric assay for horseradish peroxidase activity based on the linkage of conjugated system formed by oxidative decarboxylation

Horseradish peroxidase (HRP)-catalyzed oxidation of 2,2-bis[3-acethylfilicinic acid-5-yl]acetic acid (BAFA, 4) produces Dehydro-3,3’-diacetyl-5,5’-methylenedifilicinic acid (DDMF, 3). A new photometric hydrogen donor (4) for peroxidase (POD)-catalyzed oxidation was demonstrated to be potentially useful for spectrocolorimetric and spectrofluorometric determination of HRP. Our developed colorimetric (absorption at 483nm) and fluorometric (emission at 529nm) systems both gave a linear calibration curve for HRP (y=0.0025×+0.0237; R2=0.9997, and y=0.241×+3.194; R2=0.9914, respectively) in the same concentration range of 9.1×10−8–1.1×10−6nmol/L. The calculated Km and Vmax values of 5.10×10−4M and 5.13×10−6M/min, respectively. The results indicate that the quantification of HRP using BAFA 4 as hydrogen donor is possible.

A novel ultrasensitive competition strategy for electrochemical and colorimetric cytosensing of acute leukemia cells

Selective and reliable method is of vital importance for acute leukemia detection. In this work, a robust competitive electrochemical and colorimetric cytosensing platform for detection of acute leukemia cells was developed with high sensitivity, selectivity, acceptable rapidity and excellent extensibility. The joint effects of the high catalytic activity of horseradish peroxidase (HRP)-mimiking DNAzyme toward H2O2 reduction and the excellent competing reaction contributed to the greatly enhanced sensitivity. Such a dual mode sensing strategy allows for ultrasensitive acute leukemia cytosensing with a detection limit as low as ~4 cells and a wide linear response range from 102 to 107 cells per mL. This electrochemical and colorimetric cytosensing approach holds great promise as a new point-of-care diagnostic tool for early detection of human acute leukemia and may be readily expanded to multiplex cytosensing of other cancer cells.

Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.

Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

Acidic horseradish peroxidase activity abolishes genotoxicity of common dyes

The aim of this study was to investigate the impact of dyes on DNA before and after enzymatic decolorization by acidic horseradish peroxidase (HRP-A). The comet assay is easy and feasible method widely used to measure DNA damage and repair. The medium-throughput comet assay was employed for assessment of genotoxic effects of 8 dyes in BEAS-2B cells. We have incorporated a digestion with bacterial endonuclease (formamidopyrimidine DNA glycosylase, FPG) to detect oxidized bases in the case of single and double azo dyes, Orange II (OR2) and Amido Black 10B (AB), respectively. This allowed detection 8-oxo-7,8-dihydroguanine, one of most abundant oxidized bases in nuclear DNA. In the case of AB there was no indication of DNA damage, either strand brakes or FPG-sensitive sites before and after decolorization. The OR2 induced DNA damage (in terms of percentage of DNA in comet tails). Also, the frequency of FPG-sensitive sites increased with OR2 concentration. After decolorization no DNA damaging effects was seen at all. The interaction studies of OR2 and AB, before and after decolorization, with calf thymus DNA has been investigated by absorption and fluorescence spectroscopy. The results provide support for the idea that in some cases enzymatic decolorization contributes to lower genotoxicity potential.

Influence of NOM and SS on the BPA removal via peroxidase catalyzed reactions: Kinetics and pathways

Abstract In the present study, horseradish peroxidase (HRP) was employed to removal bisphenol A (BPA) with the presence of natural organic matter (NOM) and suspended solids (SS). Humic acid (HA) and kaolin have been selected as the typical NOM and SS in the experiment. According to the results, HA has shown a very interesting performance, which has both inhibitory and facilitation effects on BPA removal. For the low enzyme concentration (20 u/L and 50 u/L), small amount of HA could facilitate the BPA removal, while large amount of HA could inhibit the BPA removal. For the higher enzyme concentration (100–300 u/L), the addition of HA reduced the removal transformation of BPA and the inhibition effect of HA raised with the increase of HA concentrations. The facilitation nature of HA on enzyme catalytic processes can be attributed to the mitigation of enzyme inactivation, which has been proven by HRP inactivation rate. The inhibitory effect could be caused by the inhibition effect of HA on the enzyme activity, which has been suggested by both kinetic study and enzyme inactivation analysis. Kaolin showed a negative effect on BPA removal by HRP catalyzed reactions due to the inhibition effect of kaolin on HRP activity. The results have important implications for the engineering design by employing HRP in water and waste water treatment.

Proteinase K activity determination with β-galactosidase as sensitive macromolecular substrate

Proteinase K from Engyodontium album (proK) is a relatively unspecific serine endopeptidase which is known to attack proteins yet in their native states. If the attacked protein is an enzyme, even a partial hydrolysis by proK may lead to an inactivation of the enzyme, which can be monitored by measuring the loss of catalytic activity of the attacked enzyme. E. coli β-galactosidase (β-Gal) was used in this work as such enzyme. It was found to be a convenient and sensitive macromolecular model substrate for comparing the “native protein-attacking ability” of free and immobilized proK at pH = 7.0 and 23 °C. The β-Gal activity was measured spectrophotometrically with o-nitrophenyl-β-galactopyranoside. Reproducible proK determinations were possible for as little as 4.3 ng proK by using a proK analyte solution of 10 nM. Compared to free proK, immobilized proK was much less efficient in inactivating β-Gal, most likely due to a decreased mobility of immobilized proK and a restricted accessibility of β-Gal to the active site of proK. Worth noting is, that under conditions at which β-Gal was completely inactivated by proK, the activity of hen egg lysozyme, horseradish peroxidase, or Aspergillus sp. glucose oxidase remained unaltered.

Glucose Oxidase and Horseradish Peroxidase Like Activities of Cuprous Oxide/Polypyrrole Composites

Cu2O/Ppy composites coated linen texture (LT) papers possess enzyme activities of glucose oxidase (GOx) and horseradish peroxidase (HRP) in alkaline solution (0.5M NaOH). At the potential of 0V vs. Ag/AgCl reference electrode, H2O2 was produced through oxidation of glucose on the functional LT paper electrode in the presence of O2. The as-produced H2O2 was reduced on the electrode at the potential of −0.57V vs. Ag/AgCl reference electrode. Cyclic voltammograms (CVs) reveal that Cu(I)/Cu(II), Cu(II)/Cu(I), and Cu(I)/Cu transitions occur at ca. 0, −0.57, and −0.85V respectively. The electrode allows detections of glucose and H2O2 down to 0.16 and 0.41mM, respectively. The high catalytic activity of the Cu2O/Ppy composites is attributed to the stable coatings of Ppy and the high-index facets of Cu2O octahedra and microflowers. Having advantages of a low onset potential (ca. −0.02V), high sensitivity (28μAmM−1cm−2), and selectivity, the stable and durable LT paper electrode has been validated for the quantitation of glucose in blood samples, showing its great potential for monitoring blood glucose levels.

Real-time and in situ enzyme inhibition assay for the flux of hydrogen sulfide based on 3D interwoven AuPd-reduced graphene oxide network

A highly sensitive enzyme inhibition analytical platform was established firstly based on paper-supported 3D interwoven AuPd-reduced graphene oxide (rGO) network (NW) for real-time and in situ analysis of H2S released from cancer cells. The novel paper working electrode (PWE) with large electric conductivity, effective surface area and unusual biocompatibility, was fabricated via controllably assembling rGO and AuPd alloy nanoparticles onto the surface of cellulose fibers and into the macropores of paper, which was employed as affinity matrix for horseradish peroxidase (HRP) loading and cells capture. It was the superior performances of AuPd-rGO-NW-PWE that made the loaded HRP exhibit excellent electrocatalytic behavior to H2O2, bring the rapid enhancement of current response. After releasing H2S, the current response would be obviously decreased due to the efficient inhibition effect of H2S on HRP activity. The inhibition degree of HRP was directly proportional to the amount of H2S, and so, the flux of H2S released from cells could be recorded availably. Thus, this proposed enzyme inhibition cyto-sensor could be applied for efficient recording of the release of H2S, which had potential utility to cellular biology and pathophysiology.

Amperometric inhibitive biosensor based on horseradish peroxidase-nanoporous gold for sulfide determination.

As a well-known toxic pollutant, sulfide is harmful to human health. In this study, a simple and sensitive amperometric inhibitive biosensor was developed for the determination of sulfide in the environment. By immobilizing nanoporous gold (NPG) on glassy carbon electrode (GCE), and encapsulating horseradish peroxidase (HRP) onto NPG, a HRP/NPG/GCE bioelectrode for sulfide detection was successfully constructed based on the inhibition of sulfide on HRP activity with o-Phenylenediamine (OPD) as a substrate. The resulted HRP/NPG/GCE bioelectrode achieved a wide linear range of 0.1–40 μM in sulfide detection with a high sensitivity of 1720 μA mM−1 cm−2 and a low detection limit of 0.027 μM. Additionally, the inhibition of sulfide on HRP is competitive inhibition with OPD as a substrate by Michaelis-Menten analysis. Notably, the recovery of HRP activity was quickly achieved by washing the HRP/NPG/GCE bioelectrode using differential pulse voltammetry (DPV) technique in deaerated PBS (50 mM, pH 7.0) for only 60 s. Furthermore, the real sample analysis of sulfide by the HRP/NPG/GCE bioelectrode was achieved. Based on above results, the HRP/NPG/GCE bioelectrode could be a better choice for the real determination of sulfide compared to inhibitive biosensors previously reported.

Photocatalytic Reactive Oxygen Species Formation by Semiconductor-Metal Hybrid Nanoparticles. Toward Light-Induced Modulation of Biological Processes.

Semiconductor-metal hybrid nanoparticles manifest efficient light-induced spatial charge separation at the semiconductor-metal interface, as demonstrated by their use for hydrogen generation via water splitting. Here, we pioneer a study of their functionality as efficient photocatalysts for the formation of reactive oxygen species. We observed enhanced photocatalytic activity forming hydrogen peroxide, superoxide, and hydroxyl radicals upon light excitation, which was significantly larger than that of the semiconductor nanocrystals, attributed to the charge separation and the catalytic function of the metal tip. We used this photocatalytic functionality for modulating the enzymatic activity of horseradish peroxidase as a model system, demonstrating the potential use of hybrid nanoparticles as active agents for controlling biological processes through illumination. The capability to produce reactive oxygen species by illumination on-demand enhances the available peroxidase-based tools for research and opens the path for studying biological processes at high spatiotemporal resolution, laying the foundation for developing novel therapeutic approaches.

Amino-functionalized graphene quantum dots based ratiometric fluorescent nanosensor for ultrasensitive and highly selective recognition of horseradish peroxidase

Herein, a ratiometric fluorescent nanosensor for ultrasensitive and highly selective recognition of horseradish peroxidase (HRP) was reported for the first time, by employing amino-functionalized graphene quantum dots (af-GQDs) as the reference fluorophore and 2,3-diaminophenazine (DAP) as the specific response signal. DAP was the oxidation product of o-phenylenediamine (OPD) that acted as the cosubstrate of HRP. Upon the addition of HRP, OPD could be catalytically oxidized to DAP, then the fluorescence intensity corresponding to DAP at 553nm increased dramatically with a simultaneous fluorescence quenching of af-GQDs at 440nm, resulting in a ratiometric fluorescent nanosensor toward HRP. This ratiometric fluorescent nanosensor exhibited a broad linear range and excellent sensitivity toward HRP detection. The fluorescence intensity ratio of DAP to af-GQDs linearly increased with the increasing of HRP concentration in the range of 2 fM–800 fM (0.02μUmL−1–8μUmL−1) with a detection limit down to 0.21 fM (2.1nUmL−1). The present ratiometric fluorescent nanosensor also showed excellent selectivity for HRP over some amino acids, nucleotides, and other heme-containing proteins. The enzymatic activity of HRP was further evaluated by quantitatively calculating the enzymatic velocity and Michaelis−Menten kinetic parameter.

Fabrication of Lignosulfonate Vesicular Reverse Micelles to Immobilize Horseradish Peroxidase

Sodium lignosulfonate reverse micelles (SLRMs) with vesicular structure were prepared by self-assembling in ethanol–water media and applied to encapsulate horseradish peroxidase (HRP). Results showed that sodium lignosulfonate (SL) could not form SLRMs until the ethanol content reached 63% when its initial concentration was 7.5 g L–1. Owing to strong electrostatic repulsion, solid spherical SLRMs gradually swelled to stable vesicular structures with an average size of 240 nm. The shell of the SLRM thickened when NaCl was added to screen the electrostatic interaction. HRP can be effectively encapsulated while retaining its activity in the hydrophilic core of a SLRM. When hydrogen peroxide was added to initiate the catalytic activity of HRP, SL molecules would be polymerized and the structure of SLRMs would be fixed. Furthermore, HRP immobilized in polymerized SLRMs showed high activity at a more acidic pH of 4 and at a lower optimal temperature decrease of 35 °C compared to free HRP. SLRM allows enzymes su...