Modification Of Horseradish Peroxidase Research Articles

Effect of dielectric barrier discharge (DBD) plasma on the activity and structural changes of horseradish peroxidase

As an emerging nonthermal technology, cold plasma has been used to inactivate endogenous enzymes that are responsible for enzymatic browning reaction of fruits and vegetables. This study aimed to investigate the inactivation effect of dielectric barrier discharge (DBD) plasma on horseradish peroxidase (HRP), a typical plant peroxidase. The results showed that DBD plasma caused inactivation of HRP in a time-and discharge power-dependent manner. The HRP activity decreased by 32.5, 50.6, 65.5, and 75.4%, respectively, after 2, 4, 6, and 8 min of exposure to DBD plasma at 57.6 W. The efficacy of DBD plasma for HRP inactivation was enhanced on increasing the discharge power from 6.0 to 57.6 W. Intrinsic fluorescence spectra showed that DBD plasma induced obvious structural changes in HRP. DBD plasma also caused fragmentation and carbonylation of HRP as well as the oxidative degradation of heme, which might be due to the reactive species in plasma. After DBD plasma exposure at 43.0 W for 8 min, there was approximately an 8.4-fold increase in surface hydrophobicity of HRP. After exposure to DBD plasma, the aggregation of HRP was observed by using atomic force microscopy analysis. In conclusion, DBD plasma causes structural changes and chemical modification of HRP, which may be responsible for the loss of enzymatic activity. These data contribute to the application of cold plasma in the control of enzymatic browning of food products during processing and storage.

Carbon felt-based bioelectrocatalytic flow-through detectors: Highly sensitive amperometric determination of H 2O 2 based on a direct electrochemistry of covalently modified horseradish peroxidase using cyanuric chloride as a linking agent

Horseradish peroxidase (HRP) was chemically modified using cyanuric chloride (CC) as a linking agent onto a carbon felt (CF), which is a microelectrode ensemble of micro carbon fiber (>7 μm, diameter) with a random three-dimensional structure. The resulting HRP-modified CF (HRP-ccCF) exhibited well-defined redox waves based on the HRP heme Fe III/Fe II redox couple at −0.23 V vs. Ag/AgCl (at pH 7.0), while the HRP-adsorbed CF (HRP-CF) showed no apparent redox couple in the same potential range, indicating that the chemical modification of HRP via CC facilitated the direct electron transfer (DET) between HRP and CF. The apparent heterogeneous electron transfer rate constant k s was estimated to be 35 s −1. Cyclic voltammetry and electrochemical impedance spectroscopy revealed that the interfacial properties (i.e., structure, morphology of enzyme-layer) of covalently modified HRP (HRP-ccCF) and physically adsorbed HRP (HRP-CF) are different, resulting in the difference in the electron transfer properties. The HRP-ccCF was successfully used as a working electrode unit in bioelectrocatalytic flow-through detector for highly sensitive amperometric determination of H 2O 2. Under the optimized conditions (i.e., applied potential, 0 V vs. Ag/AgCl; carrier flow rate, 3.25 ml/min; and carrier pH 7.0), the cathodic peak current of H 2O 2 linearly increased up to 3 μM (sensitivity, 1.94 μA/μM; the detection limit, 0.08 μM [S/N = 3]) with sample through-put of ca. 90 samples/h.

Aggregation Behavior of Giant Amphiphiles Prepared by Cofactor Reconstitution

We report on biohybrid surfactants, termed "giant amphiphiles", in which a protein or an enzyme acts as the polar head group and a synthetic polymer as the apolar tail. It is demonstrated that the modification of horseradish peroxidase (HRP) and myoglobin (Mb) with an apolar polymer chain through the cofactor reconstitution method yields giant amphiphiles that form spherical aggregates (vesicles) in aqueous solution. Both HRP and Mb retain their original functionality when modified with a single polystyrene chain, but reconstitution has an effect on their activities. In the case of HRP the enzymatic activity decreases and for Mb the stability of the dioxygen myoglobin (oxy-Mb) complex is reduced, which is probably the result of a disturbed binding of the heme in the apo-protein or a reduced access of the substrate to the active site of the enzyme or protein.

Effect of the covalent modification of horseradish peroxidase with poly(ethylene glycol) on the activity and stability upon encapsulation in polyester microspheres

Encapsulation of proteins in polyester microspheres by coacervation methods frequently causes protein inactivation and aggregation. Furthermore, an often-substantial amount of the encapsulated proteins is released within the first 24h from the microspheres. To overcome these problems poly(ethylene glycol) (PEG) was employed as excipient and protein-modifying agent. The model protein horseradish peroxidase (HRP) was chemically modified or co-lyophilized with PEG of differing molecular weights, namely PEG5000, PEG20000, and PEG40000. The lyophilized preparations were encapsulated in poly(D,L-lactide-co-glycolic) acid (PLGA) microspheres by a coacervation method. Covalent modification of HRP with PEG increased the encapsulation efficiency (EE) from 83% to about 100% while PEG when used as an excipient reduced the EE. Encapsulation caused aggregation of ca. 5% of non-modified HRP and the residual specific activity was only 57%. Covalent modification with PEG reduced HRP aggregation to less than 1% and improved its residual activity to more than 95%. When PEG was used as excipient similar results were found with respect to a reduction in encapsulation-induced aggregation, but no more than 80% of residual activity was obtained even for the best formulation after encapsulation. It was also found that covalent modification of HRP with PEG substantially reduced the unwanted initial “burst” release observed during the initial 24h of in vitro release from about 70% to 23%. Furthermore, HRP activity and stability were also improved during in vitro release for HRP-PEG conjugates. The data show that covalent modification of proteins with PEG might be useful to improve protein stability during coacervation encapsulation and subsequent release as well as to increase EE and reduce the burst release.

Polypeptide Point Modifications with Fatty Acid and Amphiphilic Block Copolymers for Enhanced Brain Delivery

There is a tremendous need to enhance delivery of therapeutic polypeptides to the brain to treat disorders of the central nervous system (CNS). The brain delivery of many polypeptides is severely restricted by the blood-brain barrier (BBB). The present study demonstrates that point modifications of a BBB-impermeable polypeptide, horseradish peroxidase (HRP), with lipophilic (stearoyl) or amphiphilic (Pluronic block copolymer) moieties considerably enhance the transport of this polypeptide across the BBB and accumulation of the polypeptide in the brain in vitro and in vivo. The enzymatic activity of the HRP was preserved after the transport. The modifications of the HRP with amphiphilic block copolymer moieties through degradable disulfide links resulted in the most effective transport of the HRP across in vitro brain microvessel endothelial cell monolayers and efficient delivery of HRP to the brain. Stearoyl modification of HRP improved its penetration by about 60% but also increased the clearance from blood. Pluronic modification using increased penetration of the BBB and had no significant effect on clearance so that uptake by brain was almost doubled. These results show that point modification can improve delivery of even highly impermeable polypeptides to the brain.

Anthraquinone 2-carboxylic acid as an electron shuttling mediator and attached electron relay for horseradish peroxidase

Horseradish peroxidase (HRP)-modified electrodes are among the most frequently used detectors for measuring the amount of H 2O 2 as well as organic hydroperoxides. The major requirement for the construction of HRP-modified electrodes is to establish a fast and efficient electron transfer contact between the electrode surface and the redox center of peroxidase. To establish such a communication, we examined anthraquinone 2-carboxylic acid (AQ) as a novel electron shuttling mediator and attached electron relay for HRP. Cyclic voltammetric results showed that dissolved AQ molecules are able to shuttle electrons between the heme site of HRP and the glassy carbon electrode. Covalent attachment of AQ molecules to the lysine residues of HRP established a direct electrical communication between the modified enzyme and the conventional electrode. In both cases, the glassy carbon electrode exhibited high current responses to H 2O 2. Moreover, the chemical modification of HRP with AQ enhanced the catalytic activity of the enzyme by 11%.

Chemical modification of horseradish peroxidase with several methoxypolyethylene glycols

The chemical modification of e-NH2 lysine residues of horseradish peroxidase (E.C. 1.11.1.7) with several mPEG was carried out. The modified enzymes were studied through Chromatographic and electrophoretic methods; the extent of mPEG linking was determined using1H-NMR. Peroxidase, modified with mPEG ranging from 350 to 5000, activated with 4-nitrophenylchloroformate (mPEGpn), showed a better thermal stability than the native, but there was no correlation between the length of the polymer adduct and the improvement. The enzyme was modified with mPEG (5000) activated by cyanuric chloride (mPEGcc). The number of modified lysine increased, but the thermal behavior of mPEGcc peroxidase was similar to those of mPEGpn enzymes. In all cases, the modification did not markedly change the stability in organic solvents.

Reactivities of Organic-Phase Biosensors. 1. Enhancement of the Sensitivity and Stability of Amperometric Peroxidase Biosensors Using Chemically Modified Enzymes

Amperometric organic- and aqueous-phase peroxide biosensors were prepared with native and N-hydroxysuccinimide ester (NHS)-modified horseradish peroxidase (HRP). The e-amino functions of the free lysine residues of HRP were selectively modified with homobifunctional suberic acid bis(N-hydroxysuccinimide ester) (SA-NHS) or ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) (EG-NHS). The biosensors were based on electrostatic complexation of the HRP variants with an osmium bis(bipyridyl)poly(4-vinylpyridine) polymer. The enzyme electrodes were operated in both aqueous (0.1 M phosphate buffer, pH 7.0) and organic (90% CH3CN) phases, for the detection of hydrogen peroxide and methyl isothiocyanate. Kinetic analysis of the steady-state amperometry data showed that chemical modification of HRP brought up to a ∼5-fold enhancement of the biosensor sensitivity for H2O2 and an improvement in both sensor stability and organotolerance. Also, NHS modification of HRP results in a 3-fold extension of the rang...

Modification of horseradish peroxidase with bifunctional n-hydroxysuccinimide esters: Effects on molecular stability

Horseradish peroxidase (HRP) has been chemically modified with the homobifunctional cross-linking reagents suberic acid n-hydroxysuccinimide ester (SA-NHS) and ethylene glycol bis-succinimidyl succinate (EG-NHS) yielding derivatives of native HRP with enhanced stability in a range of organic solvents. Modification did not cause any loss of activity. It has been shown that these modification reagents react specifically with the ϵ-amino groups of the six lysine residues of HRP. Stability increased with concentrations of EG-NHS up to a level of 4–5 mg ml −1. A marked difference was observed in the tryptophan fluorescence profiles of native and the EG-NHS peroxidases upon thermoinactivation at 65°C. Both modified peroxidases exhibited improved resistance to the denaturant guanidine hydrochloride.

Interaction of aromatic donor molecules with horseradish peroxidase: identification of the binding site and role of heme iron in the binding and activity

The interaction of aromatic substrates with horseradish peroxidase (HRP) was studied. Chemical modification of HRP was performed using diethylpyrocarbonate (DEPC) and for the first time the amino acid involved in binding with these substrates has been identified. The kinetic parameters for this interaction have been calculated and the role of heme iron in the oxidation of aromatic substrates by HRP has been discussed.

Enzyme-antibody conjugation by a heterobifunctional reagent and its application in enzyme-linked immunosorbent assay (ELISA) for the detection of hepatitis B surface antigen

The heterobifunctional reagent 3-(2-pyridyl-dithio)propionate (SPDP) was used to prepare defined conjugates composed of horseradish peroxidase (HRP) and goat anti-HBs IgG. The modification of HRP and IgG with SPDP was dependent on both the SPDP: protein molar ratio and the pH of the buffer. Conjugates were separated by a single affinity Chromatographic step using concanavalin A-Sepharose 4B equilibrated with 0.1 M Tris-HCl buffer, pH 7.4, containing 0.5 M KCl and 1 mM EDTA. The conjugate was eluted with 10 or 100 mM α-methyl- d-mannoside and appropriate pools were made reflecting various HRP/IgG molar ratios. Each pool was examined for performance in an enzyme-linked immunosorbent assay for HBsAg. Conjugate composed of an HRP/IgG molar ratio of 2.5–4 yielded the greatest sensitivity.