Enzymatic Activity Of Horseradish Peroxidase Research Articles

Photoswitching enzymatic activity of horseradish peroxidase by graphene oxide for colorimetric immunoassay

In contrast to the conventional means that the activity of horseradish peroxidase (HRP) is initiated and terminated by the additives of peroxides and strongly acidic stop solutions, this study demonstrates that the enzymatic activity of HRP is switched through the visible light irradiated graphene oxide (GO). And this visible light driven activity of HRP can realize time-precise control without the aids of peroxides (typically H2O2) and acidic stop solutions. The superoxide anions (O2•−) and photogenerated holes (h+) produced by the photo irradiated GO are responsible for activating HRP and the subsequent oxidation of the typical substrates, i.e., 3, 3′, 5, 5′-tetramethylbenzidine (TMB) and 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). It is also validated that the photoswitchable HRP-GO mixture can act as an efficient signal reporter of bioassays by taking the sandwich immunoassay of alpha-fetoprotein (AFP) as an example. The AFP can be detected sensitively and selectively in the linear range from 0.2 fg/mL to 1.0 ng/mL, with a very low detection limit of 0.1 fg/mL. Advantages of the photoswitchable HRP-GO mixture include high catalytic ability, precise time control, and free of additionally harmful reagents.

Improving the thermostability of horseradish peroxidase by incorporating into water-immiscible coacervates

Looking for novel matrix materials for encapsulation of enzymes based on water immiscible coacervates prepared by reaction of negatively charged hyaluronic acid and a positively charged recombinant mussel adhesive protein containing tyrosine residues was the subject of the investigation of this work. The results of experimental study of the thermostability of horseradish peroxidase (HRP) by means its encapsulation in these coacervates at temperature from 30 to 95 o C is presented in this paper. The Michaelis-Menten equation was applied to analyze of the enzymatic activity of HRP. The kinetic parameters were interpreted using a Lineweaver-Burk plot. According to the data obtained, Michaelis-Menten parameters, K M and K Cat , interpreted from the Lineweaver- Burk plots, were 0.271 mM and 2265 s -1 for the free HRP and 0.325 mM and 2158 s -1 for the rMAP/HA coacervate, containing HRP, respectively, which indicate that the enzyme did not lose its activity during the coacervate formation. It was founded that the free enzyme began to lose activity above 40 o C, while the encapsulated HRP remained stable to 85 o C. The encapsulated HRP lost only 18% and 25% of activity at temperature of 90 and 95 o C, respectively, while as free HRP loses all its initial activity, although they show similar activity at room temperature. Keywords: encapsulation, hyaluronic acid, coacervate, recombinant mussel adhesive protein

Enzyme structure and function protection from gastrointestinal degradation using enteric coatings

Bovine Serum Albumin (BSA) and Horseradish Peroxidase (HRP) have been encapsulated within microparticulated matrices composed of Eudragit RS100 by the water-in-oil-in-water double emulsion solvent evaporation method. Good encapsulation efficiencies were achieved for BSA and HRP, 88.4 and 95.8%, respectively. The stability of the loaded proteins was confirmed by using circular dichroism and fluorescence. The gastroresistance of the protein-loaded microparticles was evaluated under simulated gastric conditions demonstrating the preservation of the structural integrity of the proteins loaded inside the particles. The enzymatic activity of HRP after being released from the enteric microparticles was evaluated by using the peroxidase substrate, revealing that the released enzyme preserved its 100% function. The high drug loadings achieved, reduced cytotoxicity and efficient gastric protection point out towards the potential use of those carriers as oral delivery vectors of therapeutic proteins offering a more controlled targeted release in specific sites of the intestine and an enhanced gastrointestinal absorption.

Photoelectrochemical TiO2 nanotube arrays biosensor for asulam determination based on in-situ generation of quantum dots

Inspired by the photoelectrochemical (PEC) properties of TiO2 nanotubes arrays (TNA) and their application as a super vessel for immobilizing biomolecules, we constructed an inhibition-effect PEC biosensor for determination of asulam based on the in-situ generation of CdS quantum dots (QDs) on TNA using an enzymatic reaction. Horseradish peroxidase (HRP) enzyme was covalently assembled on the inner-wall of TNAs, which exhibited good electrochemical and catalytic properties. In the mixture solution containing H2O2, CdY and S2O32-, HRP enzyme in TNAs catalyzed H2O2 reduce S2O32- to S2-. The generated S2- reacted with CdY to form CdS QDs in situ on the TNAs, improving the PEC performance of TNA under visible light irradiation. The photocurrent would decrease after addition of asulam due to its inhibitory effect towards HRP enzyme activity. Under the optimal experimental conditions, the constructed PEC TNA/HRP biosensor exhibited a satisfying linear range (0.02–2.0 ng mL−1), low limit of detection (4.1 pg mL−1) and good selectivity towards asulam determination, and has been successfully applied for the analysis of real environmental water samples with good accuracy of the recoveries ranged from 90% to 114%.

Enzyme activity of horseradish peroxidase in surfactant-free microemulsions

In the present contribution, we investigated the influence of the structuring of surfactant-free microemulsions (SFME) (water/1-propanol/limonene and water/tert-butanol/limonene) on the enzyme activity of horseradish peroxidase (HRP). To this purpose, the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) with hydrogen peroxide was chosen as a model reaction. Enzymatic activities in SFMEs of varying compositions were investigated by UV–Vis spectroscopy and compared to the enzyme activity in pure buffer solution. Dynamic light, small-angle-X-ray scattering and conductivity measurements were performed in order to obtain structural information on the used SFMEs.Findings presented in this study revealed that the ability of short-chain alcohols to form mesostructures (aqueous aggregates in oil) has a crucial effect on the enzyme activity in SFMEs. Mesoscale structuring with 1-propanol (NPA) was found to be more pronounced than for the more hydrophobic tert-butanol (TBA). It was concluded that the most pronounced mesoscale-structured SFMEs lead to the highest enzymatic activities.

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.

Injectable hydrogel systems crosslinked by horseradish peroxidase

Hydrogels are widely used as reservoirs in drug delivery and scaffolds for tissue engineering. In particular, injectable hydrogel systems, which are formed by physical, chemical, or enzyme-mediated crosslinking reactions in situ, offer the advantages of minimal invasiveness, ease of application, and void-filling property. Examples of these hydrogels are provided in the first part of this paper. In the second part, hydrogels that are formed by the enzymatic activity of horseradish peroxidase (HRP) are highlighted. HRP catalyzes the crosslinking reaction of polymer-phenol conjugates in the presence of hydrogen peroxide (H2O2), resulting in hydrogels with tunable gelation rate and crosslinking density. The catalytic mechanism of the HRP-mediated crosslinking reaction is discussed in detail, and the recent biomedical applications of the HRP-crosslinked hydrogels are described. Lastly, the concerns associated with HRP-mediated crosslinking and the future outlook of HRP-crosslinked hydrogels are addressed.

Purpose-Oriented Antibody Libraries Incorporating Tailored CDR3 Sequences

The development of in vitro antibody selection technologies has allowed overcoming some limitations inherent to the hybridoma technology. In most cases, large repertoires of antibody genes have been assembled to create highly diversified libraries allowing the isolation of antibodies recognizing virtually any antigen. However, these universal libraries might not allow the isolation of antibodies with specific structural properties or particular amino acid contents that are rarely found in natural repertoires. Purpose-oriented libraries specially designed to incorporate desired characteristics have been successfully used. However, the workload required for library construction has limited the attractiveness of this approach compared to the use of large universal libraries. We have developed an approach to capture synthetic or natural diversity into the complementarity determining regions 3 (CDR3) of human antibody repertoires using Type IIS restriction enzymes. In this way, we generated several libraries either biased in amino acid content or towards long CDRH3 loops. The latter were successfully used to identify antibodies inhibiting the enzymatic activity of horseradish peroxidase, whereas libraries enriched in histidines allowed for the isolation of antibodies binding to human Fc in a pH-dependent manner. These libraries indicate that tailored diversification of CDR3 is sufficient to generate purpose-oriented libraries and isolate antibodies with uncommon properties.

Enhanced catalytic activity of enzymes interacting with nanometric titanate nanosheets

Enzymatic activity of horseradish peroxidase (HRP) at diluted conditions is highly increased under the presence of nanometric titanate nanosheets (TNS).

Poly(methyl methacrylate) particles for local drug delivery using shock wave lithotripsy: In vitro proof of concept experiment.

To leverage current local drug delivery systems methodology, there is vast use of polymeric particles serving as drug carriers to assure minimal invasive therapy with little systemic distribution of the released drug. There is an increasing interest in poly(methyl methacrylate) (PMMA) serving as carriers in drug delivery. The study aims to develop PMMA carriers for localized drug delivery and release system, combining innovative biomaterial technology and shock wave lithotripsy (SWL), and to study the effect of SWL on various concentrations of PMMA particles. We prepared PMMA particles that contain horseradish peroxidase (HRP) using a double emulsion technique, and investigated the mechanism of in vitro drug release from the carriers following exposure to SWL. We investigated the correlation between production method modifications, concentrations of the carriers subjected to SWL, and shock wave patterns. We successfully produced PMMA particles as drug carriers and stimulated the release of their contents by SWL; their polymeric shell can be shattered externally by SWL treatment, leading to release of the encapsulated drug. HRP enzyme activity was maintained following the encapsulation process and exposure to high dose of SWL pulses. Increased shock wave number results in increased shattering and greater fragmentation of PMMA particles. The results demonstrate a dose-response release of HRP; quantitation of the encapsulated HRP from the carriers rises with the number of SWL. Moreover, increased concentration of particles subjected to the same dose of SWL results in a significant increase of the total HRP release. Our research offers novel technique and insights into new, site-specific drug delivery and release systems.

Horseradish peroxidase enzyme immobilized graphene quantum dots as electrochemical biosensors.

Green colour emitting graphene quantum dots (GQDs) are prepared by a simple acid reflux reaction of graphene oxide (GO) produced using a modified Hummer's method. Structural and morphological characterizations of such GQDs are performed using spectroscopic (FTIR, UV-vis and photoluminescence) and microscopic (transmission electron microscopy) techniques. These studies reveal the formation of stable, uniform spherical particles of GQDs which emit a green colour and possess surface functional moieties such as epoxide, hydroxyl (-OH) and carboxyl (-COOH) groups. Further, the possibility of immobilizing biomolecules on GQDs using these surface active functional groups is explored. As an example, an enzyme namely horseradish peroxidase (HRP) is shown to be anchored on these GQDs using a coupling reaction between an acid and amine leading to the formation of a peptide amide bond. Enzymatic activity of HRP is investigated by simply drop-casting HRP-immobilized GQDs onto a glassy carbon electrode. Electrochemical studies clearly reveal the formation of a well-defined redox peak and the dependence of redox peak current on scan rate suggests that the HRP enzyme is anchored onto the electrode, surface confined and exhibits a direct electron transfer process that is predominantly controlled by a diffusion process. These HRP-functionalized GQDs are used as a sensing platform for hydrogen peroxide detection. This particular electrochemical biosensor shows the sensitivity values of 0.905 and 7.057μA/mM and detection limits of ~530nM and 2.16μM along with a fast response time of ~2-3s.

Controlling Local Substrate Concentrations in Multi-Enzyme Complexes

In nature we find many examples of bifunctional enzymes that effectively process cascade reactions. For example, the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) couples two active sites on the same polypeptide structure via an electrostatic patch. Interactions between the negatively charged cascade intermediate, dihydrofolate, and the positively charged enzyme surface create an environment for bounded diffusion between active sites, thus promoting substrate channeling. This is a useful example from which we can draw design inspiration for synthetic enzyme cascades for multi-step reactions in biofuel cells. To this end, our research group is designing new nanostructured multi-enzyme complexes to study the effects of cascade structure on reaction kinetics. Our overall goal is to turn our understanding of these relationships into a generalized set of design rules that can be used to engineer optimized cascade catalysis. The first step is to investigate interactions between multi-enzyme scaffolds and cascade substrates. The TS-DHFR example suggests that substrate-scaffold interactions are important and can be beneficial to cascade catalysis. Here, we demonstrate that DNA scaffolds can enhance the kinetics of assembled enzymes and that these enhancements are related to the binding energy of the substrate and DNA scaffold. A model system of horseradish peroxidase (HRP) with phenolic substrates and a triangular DNA scaffold (sides ~ 25 nm in length) showed increased enzyme activity with one, two, and three HRPs assembled on the scaffold over freely diffusing HRP modified with short single stranded DNA. Interestingly, the enhancements in activity mimicked the Sabatier principle and a plot of the kinetic enhancement as a function of substrate-DNA binding energy followed the trends of a volcano plot commonly described in heterogeneous catalysis. No enhancement in enzyme activity was observed when the binding of the substrate to the scaffold was weak (kd = 10 mM). No enhancement occurred when binding was strong (kd = 4 mM). With intermediate binding (kd = 100 – 1000 mM) the enhancement in enzyme activity of HRP assembled on a DNA scaffold over freely diffusing enzymes was significant. We hypothesize that the enhancement was due to an increase in local concentration of the substrate resulting from substrate-DNA interactions. We confirm this hypothesis by demonstrating control over the apparent Michaelis constant of HRP-DNA nanostructures by tuning the interactions between substrates and DNA scaffold. These findings represent an important first step in designing multi-enzyme complexes and demonstrate that interactions between substrates and the scaffolds must be considered when engineering such structures. Our current work is focused on extending these findings to controlling the local concentration of enzyme co-factors used in biofuel cell anodes for multi-step oxidation cascades.

Enzymatic etching of gold nanorods by horseradish peroxidase and application to blood glucose detection.

Gold nanorods (AuNRs) have become some of the most used nanostructures for biosensing and imaging applications due to their plasmon-related optical response, which is highly sensitive toward minute changes in the AuNR aspect ratio. In this context, H2O2 has been used to trigger the chemical etching of AuNRs, thereby inducing a decrease of their aspect ratio. However, special conditions and relatively high concentrations of H2O2 are usually required, preventing the applicability of the system for biodetection purposes. To overcome this limitation we have introduced a biocatalytic species, the enzyme horseradish peroxidase (HRP) that is able to induce a gradual oxidation of AuNRs in the presence of trace concentrations of H2O2. Interestingly, the presence of halide ions has also been found to be essential for this process. As a consequence, other enzymatic reactions, such as those catalyzed by glucose oxidase, can be easily coupled to HRP activity, allowing the detection of different amounts of glucose. On the basis of these findings, we developed a highly sensitive and simple colorimetric assay that can be read out by the naked eye and allows the detection of physiological glucose concentrations in human serum.

Enzymatic Assay for Cu(II) with Horseradish Peroxidase and Its Application in Colorimetric Logic Gate

We report an ultrasensitive and colorimetric assay for Cu(II) via enzymatic amplification strategy. The enzymatic activity of horseradish peroxidase (HRP) is strongly inhibited by Cu(I), which can be used indirectly to assay Cu(II). The limit of detection (LOD) is 0.37 nM, and the detection of 20 nM Cu(II) in solution can be achieved with naked eyes. This assay can be used to construct a colorimetric logic gate.

DNA-directed immobilization of horseradish peroxidase onto porous SiO2 optical transducers

Multifunctional porous Si nanostructure is designed to optically monitor enzymatic activity of horseradish peroxidase. First, an oxidized PSi optical nanostructure, a Fabry-Pérot thin film, is synthesized and is used as the optical transducer element. Immobilization of the enzyme onto the nanostructure is performed through DNA-directed immobilization. Preliminary studies demonstrate high enzymatic activity levels of the immobilized horseradish peroxidase, while maintaining its specificity. The catalytic activity of the enzymes immobilized within the porous nanostructure is monitored in real time by reflective interferometric Fourier transform spectroscopy. We show that we can easily regenerate the surface for consecutive biosensing analysis by mild dehybridization conditions.

Supported biocatalysts for Alizarin and Eriochrome Blue Black R degradation using hydrogen peroxide

The goal of this manuscript was to study the immobilization of horseradish peroxidase (HRP) and hematin on different supports at selected conditions to be used in the removal of two synthetic dyes: Alizarin Red (AR) and Eriochrome Blue Black R (EBBR). Reuse in the target reactions was tested for the best biocatalysts. Three supports were selected: magnetite-activated carbon Fe3O4/C, activated carbon C and chitosan CS. Several reasons led to discard the use of Fe3O4/C and C as supports for hematin and HRP. CS–glutaraldehyde (GA)–HRP not only has low ability to remove EBBR, but also the enzymatic activity of HRP could not be separated from the dye adsorption on CS. However, some adsorptive functions of CS–GA were lost when HRP was supported on it.In the case of CS–GA–hematin, the most important factors for EBBR removal are dye concentration and mass of catalyst, whereas the temperature has almost no effect on the conversion of dye, considering in all cases the H2O2 concentration (0.021M) constant.There was not a marked decrease in the removal of AR even after the ninth reuse in the case of CS–GA–hematin. For EBBR removal, with CS–GA–HRP, in the second reuse the catalyst was completely deactivated, whereas with CS–GA–hematin this was found only in the fourth reuse.

Design and Preparation of a Nanoprobe for Imaging Inflammation Sites

To image inflammation sites, we developed a novel nanoparticle, hydroxylamine-containing nanoparticle (HANP), which emits an intense electron spin resonance (ESR)-signal triggered by enzymatic oxidation reaction and pH-sensitive self-disintegration. The nanoparticle was prepared from an amphiphilic block copolymer, poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-hydroxyl)aminomethylstyrene] (PEG-b-PMNT-H), which spontaneously forms a core-shell type polymeric micelle (particle diameter = ca. 50 nm) in aqueous media. Because the PMNT-H segment in the block copolymer possesses amino groups in each repeating unit, the particle can be disintegrated by protonation of the amino groups in an acidic pH environment such as inflammation sites, which is confined to the hydrophobic core of HANP. Mixing HANP with horseradish peroxidase (HRP)/H(2)O(2) mixture resulted in enzymatic oxidization of the hydroxylamines in the PEG-b-PMNT-H and converted the hydroxylamine to the stable nitroxide radical form in PEG-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene] (PEG-b-PMNT), which shows an intense ESR signal. It is interesting to note that the ESR signal increased at a greater rate under acidic conditions (pH 5.6) than that under neutral conditions (pH 7.4), although the enzymatic activity of HRP under neutral conditions is known to be much higher than that under acidic conditions. This indicates that enzymatic oxidation reaction was accelerated by synchronizing the disintegration of HANP under acidic conditions. On the basis of these results, HANP can be used as a high-performance ESR probe for imaging of inflammation sites.

Determination of selected persistent organic pollutants in wastewater from landfill leachates, using an amperometric biosensor

Landfill leachates that contain persistent organic pollutants (POPs) are a big threat to groundwater systems and are projected to have hazardous effects in the long term if proper management strategies of the landfills are not put in place by those responsible. Monitoring the levels of POPs in landfill leachates is very crucial. This work presents an amperometric biosensor for determination of selected POPs in landfill leachates. The biosensor is based on kinetic inhibition of horseradish peroxidase (HRP). The enzyme was immobilised by electrostatic attachment on a polyaniline-modified Pt electrode surface. Selected POPs inhibited HRP enzyme activity and the decrease in the enzyme activity was used to determine these environmental pollutants. Selected polybrominated diphenyl ethers (PBDEs), polybrominated biphenyls (PBBs) and polychlorinated biphenyls (PCBs) were the analytes of choice because they are commonly found in South Africa water systems. Limits of detection for the amperometric biosensor were established as 0.014, 0.018, 0.022, 0.016 and 0.019μgl−1 for BDE-100, PBB-1, PCB-1, PCB-28 and PCB-101, respectively. The HRP biosensor system gave different linear ranges for; BDE-100 (0.424–25.8μgl−1), PBB-1 (0.862–13.4μgl−1), PCB-1 (0.930–18.1μgl−1), PCB-28 (0.730–15.7μgl−1) and PCB-101 (0.930–27.1μgl−1). Inhibition studies on HRP biosensor response toward the reduction of H2O2 in the absence and presence of the selected POPs were carried out to investigate the inhibition kinetics and its mechanism. The results obtained indicated that the inhibition mechanism was competitive for PBDEs and non-competitive for biphenyls (PCBs and PBBs). The application of the biosensor was tested on wastewater samples obtained from landfill leachate for determination of selected POPs. The leachate samples were found to contain PCB-28 (0.28±0.03μgl−1) and PCB-101 (0.31±0.02μgl−1). The samples were also analysed by GC–MS as a cross-check method and the two sets of results were in close agreement.

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...