Microperoxidase-11 Research Articles

4-Aminobenzoic acid hydrazide inhibition of microperoxidase-11: Catalytic inhibition by reactive metabolites

Inhibition of human peroxidase enzymes such as myeloperoxidase or eosinophil peroxidase represents a novel therapeutic area, for which there are no current clinical therapeutics. We utilized 4-aminobenzoic acid hydrazide which was reported to be a potent irreversible inhibitor of myeloperoxidase to gain insight into the role of reactive metabolites in catalytic inhibition. In order to carry out detailed studies, we used a model peroxidase, microperoxidase-11 (MP-11). We investigated the heme spectrum of MP-11 in the presence of 4-ABAH and found that heme bleaching occurred that was irreversible. This coincided with an absence of catalytic activity. The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was able to significantly prevent inactivation of peroxidase activity, therefore, we performed ESR spin trapping studies and detected a carbonyl carbon-centered radical of 4-ABAH. In order to determine if the free radical metabolites became bound to MP-11, we performed high-resolution MALDI with elemental analysis to determine the change in elemental composition that occurred in these reactions. These masses were assigned to free radical metabolites of 4-ABAH and were not observed in reactions containing DMPO. We conclude that the 4-ABAH free radical metabolites which were bound to MP-11 were involved in the catalytic inhibition and were scavenged by DMPO.

Nano polyurethane-assisted ultrasensitive biodetection of H2O2 over immobilized Microperoxidase-11

Nanostructured polyurethane (PU) synthesized by an emulsion polymerization with narrow size distribution was employed for the first time directly as a novel matrix for enzyme immobilization to develop sensitively amperometric biosensors. When Microperoxidase-11 (MP-11) was selected as a model protein, the resulting hydrogen peroxide (H2O2) biosensor exhibited improved sensitivity of 29.6μAmM−1cm−2 with quite good response time of (1.3±0.4)s and remarkable limit of detection as low as 10pM (S/N 3) over existing protocols. A linear calibration curve for hydrogen peroxide was obtained up to 1.3μM under the optimized conditions with a relative low calculated Michaelis–Menten constant (KMapp) (1.87±0.05)μM, which indicated the enhanced enzymatic affinity of MP-11 to H2O2 via PU. The possible interferents had negligible effect on the response current and time of the prepared biosensor. Results suggest that the PU nanoparticles (PU-NPs) with good biocompatibility and sufficient interfacial adhesion hold promise as an attractive support material for construction of ultrasensitive amperometric biosensor.

Bioelectrocatalysis by Microperoxidase‐11 in a Multilayer Architecture of Chitosan Embedded Gold Nanoparticles

AbstractWe report on the redox behaviour of the microperoxidase‐11 (MP‐11) which has been electrostatically immobilized in a matrix of chitosan‐embedded gold nanoparticles on the surface of a glassy carbon electrode. MP‐11 contains a covalently bound heme c as the redox active group that exchanges electrons with the electrode via the gold nanoparticles. Electroactive surface concentration of MP‐11 at high scan rate is between 350±50 pmol cm−2, which reflects a multilayer process. The formal potential (E°′) of MP‐11 in the gold nanoparticles‐chitosan film was estimated to be −(267.7±2.9) mV at pH 7.0. The heterogeneous electron transfer rate constant (ks) starts at 1.21 s−1 and levels off at 6.45 s−1 in the scan rate range from 0.1 to 2.0 V s−1. Oxidation and reduction of MP‐11 by hydrogen peroxide and superoxide, respectively have been coupled to the direct electron transfer of MP‐11.

Direct Evidence of Covalent Immobilisation of Microperoxidase‐11 on Plasma Polymer Surfaces

AbstractThe question of whether immobilised proteins are bound covalently on plasma activated or deposited polymers has been a challenge for almost 30 years, as there has been no directly evidence to conclude unambiguously. In this paper, we report evidence that a chemical binding reaction occurred during immobilisation of microperoxidase‐11 (MP‐11) proteins on plasma polymer surfaces. Untreated polymer surfaces were not resistant to detergent cleaning and did not show any increase of S 2p component after protein immobilisation, whilst all plasma‐deposited surfaces showed large amounts of immobilised MP‐11 proteins after detergent cleaning. We conclude that the MP‐11 protein had a chemical binding reaction thorough its cysteine residues with the plasma polymer surfaces.magnified image

Direct Electrochemistry and Bioelectrocatalysis of Microperoxidase‐11 Immobilized on Chitosan‐Graphene Nanocomposite

AbstractA bioelectrochemical platform has been constructed for the direct electron transfer and biosensing purposes of microperoxidase‐11 (MP‐11) immobilized on the chitosan dispersed multilayer graphene nanocomposite. The immobilized MP‐11 at the modified gold electrode displays a well‐defined and quasireversible redox peaks, with a formal potential of −0.38 V/SCE in a buffer solution (pH 7.0). MP‐11 absorbed on the electrode surface exhibits high electrocatalytic activity toward the reduction of both oxygen and hydrogen peroxide and also shows good analytical performance for the amperometric detection of H2O2 with a linear range from 2.5 to 135 μM. These results indicate the graphene modified electrode might be used as a third generation biosensor for H2O2 detection.

Spatial organization of peptide nanotubes for electrochemical devices

A novel biosensor for hydrogen peroxide was developed by combining the known properties of microperoxidase-11 (MP11) as an oxidation catalyst, and the interesting properties of diphenylalanine peptide nanotubes (PNTs) as a supporting matrix to allow a good bioelectrochemical interface. In this case, the synthesized MP11/PNTs were immobilized onto the ITO electrode surface via layer-by-layer (LBL) deposition, using poly(allylamine hydrochloride) (PAH) as positively charged polyelectrolyte layers. The PNTs provide a favorable microenvironment for MP11 to perform direct electron transfer to the electrode surface. The resulting electrodes showed a pair of well-defined redox peaks with formal potential at about −343 mV (versus SCE) in phosphate buffer solution (pH 7). The experimental results also demonstrated that the resulting biosensor exhibited good electrocatalytic activity to the reduction of H2O2 with a sensitivity of 9.43 μA cm−2 mmol−1 L, and a detection limit of 6 μmol L−1 at the signal-to-noise ratio of 3. Moreover, we also observed that the peptides self-assembly can be influenced upon changing the pH of the solution. Alkaline solution appears to favor the packing of diphenylalanine nanotubes being closer than acidic or neutral conditions. The study proved that the combination of PNTs with MP11 is able to open new opportunities for the design of enzymatic biosensors with potential applications in practice.

Preparation of Gold Nanotube by Direct Electrodeposition for Biosensors

A simple method to fabricate gold nanotube by means of a direct electrodeposition is constructed, utilizing anodic aluminum oxide (AAO) as a template. The performances of gold nanoelectrode have been characterized with cyclic voltammetric technique and scanning electron microscope (SEM). The SEM image of as-prepared gold nanoelectrode shows that the surface of electrode was covered with honeycomb gold tube with average pore diameter 200 nm. Its cyclic voltammetric shows the peak current is 6.25 times larger than the bare Au electrode, so the new honeycomb gold nanoelectrode was obviously better than conventional gold electrode. Microperoxidase-11 (MP-11) was immobilized on the electrode and characterized with cyclic voltammetric technique. It was demonstrated that the gold nanotube not only could offer a friendly environment to immobilize MP-11, but also enhance the electron transfer ability between protein molecules and the underlying electrodes.

Rectified tunneling current response of bio-functionalized metal–bridge–metal junctions

Biomolecular bridged nanostructures allow direct electrical addressing of electroactive biomolecules, which is of interest for the development of bioelectronic and biosensing hybrid junctions. In the present paper, the electroactive biomolecule microperoxidase-11 (MP-11) was integrated into metal–bridge–metal (MBM) junctions assembled from a scanning tunneling microscope (STM) setup. Before immobilization of MP-11, the Au working electrode was first modified by a self-assembled monolayer of 1-undecanethiol (UDT). A symmetric and potential independent response of current–bias voltage ( I t / V b ) was observed for the Au (substrate)/UDT/Au (tip) junction. However, the I t / V b characteristics became potential dependent and asymmetrical after binding of MP-11 between the electrodes of the junction. The rectification ratio of the asymmetric current response varies with gate electrode modulation. A resonant tunneling process between metal electrode and MP-11 enhances the tunneling current and is responsible for the observed rectification. Our investigations demonstrated that functional building blocks of proteins can be reassembled into new conceptual devices with operation modes deviating from their native function, which could prove highly useful in the design of future biosensors and bioelectronic devices.

Microperoxidase 11: a model system for porphyrin networks and heme–protein interactions

We measured the circular dichroism (CD) and absorption spectra of the B-band region of microperoxidase 11 (MP11) as a function of temperature and peptide concentration. At micromolar concentrations, small MP11 dimers or trimers lead to excitonic coupling between low-spin and high-spin heme groups, to which the NH(2) group of the MP11 N-terminal and H(2)O are bound as a sixth ligand, respectively. These aggregates convert into monomers with hexacoordinated high-spin heme groups with increasing temperature. This transition can be described by a two-state model. Aggregation becomes more extended at 50 microM concentration and causes some B-band hyperchromism, which reflects a J-type arrangement of heme groups linked together in the aggregates formed. At near-millimolar concentration, the CD and absorption spectra of the B-band region suggest the existence of even more extended and thermally stable aggregates, which might involve mu-oxo dimers of the heme groups. The degree of aggregation at 50 and 500 microM concentration increases substantially if the sample is freed from most of its oxygen in a N(2) atmosphere. The CD spectrum of the monomeric high-spin species is reminiscent of that observed for the unfolded alkaline conformation of the intact protein. Finally, we investigated the binding of acetylmethionine (AcM) ligands to the heme at aggregation-supporting conditions (500 microM concentration). The data suggest that the ligand prevents any substantial aggregation. As a surprising result, our data reveal that AcM-MP11 complexes exhibit a high-spin/low-spin mixture, with the high-spin configuration being stabilized at high temperatures.

Control of Bioelectrocatalytic Transformations on DNA Scaffolds

The spatial organization of biomolecules on a DNA scaffold linked to an electrode leads to programmed biocatalytic transformations. This is exemplified by the electrical contacting of glucose oxidase (GOx) linked to the DNA scaffold with the electrode. A nucleic acid functionalized with a ferrocene relay unit was hybridized with the DNA scaffold at a position adjacent to the electrode, and GOx functionalized with nucleic acid units complementary to the specific domain of the DNA template was hybridized with the DNA scaffold in a position remote from the electrode. Under these conditions, ferrocene-mediated oxidation of the redox center of GOx occurred, and the effective bioelectrocatalytic oxidation of glucose was activated. Exchange of the position of GOx and the electron-mediator groups prohibited the bioelectrocatalytic oxidation of glucose. In another system, a nucleic acid-functionalized microperoxidase-11 (MP-11) and the nucleic acid-modified GOx were hybridized with the adjacent and remote sites, respectively, on the DNA scaffold associated with the electrode. In this configuration, effective MP-11-catalyzed reduction of H(2)O(2) generated by the GOx-catalyzed oxidation of glucose occurred, and the resulting bioelectrocatalytic cathodic currents were controlled by the concentration of glucose. Exchanging the positions of MP-11 and GOx on the DNA scaffold eliminated the MP-11-electrocatalyzed reduction of H(2)O(2).

A hybrid biofuel cell based on electrooxidation of glucose using ultra-small silicon nanoparticles

The ultra-small silicon nanoparticle was shown to be an electrocatalyst for the electrooxidation of glucose. The oxidation appeared to be a first order reaction which involves the transfer of 1 electron. The oxidation potential showed a low onset of −0.4 V vs. Ag/AgCl (−0.62 V vs. RHE). The particle was used as the anode catalyst of a prototype hybrid biofuel cell, which operated on glucose and hydrogen peroxide. The output power of the hybrid cell showed a dependence on the enzymes used as the cathode catalyst. The power density was optimized to 3.7 μW/cm 2 when horseradish peroxidase was replaced by microperoxidase-11 (MP-11). Comparing the output power of the hybrid cell to that of a biofuel cell indicates enhanced cell performance due to the fast reaction kinetics of the particle. The long-term stability of the hybrid cell was characterized by monitoring the cell voltage for 5 days. It appeared to that the robustness of the silicon particle resulted in more cell stability compared to the long-term performance of a biofuel cell.

Biosensor based on the biocatalysis of microperoxidase-11 in nanocomposite material of multiwalled carbon nanotubes/room temperature ionic liquid for amperometric determination of hydrogen peroxide

A novel nanocomposite material of multiwalled carbon nanotubes (MWCNTs) and room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF6) was explored and used to construct a novel microperoxidase-11 (MP-11) biosensor for the determination of hydrogen peroxide (H2O2). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to characterize the performance of the biosensor. Under the optimized experimental conditions, H2O2 could be detected in a linear calibration range of 0.5 to 7.0 x 10(-7)mol L(-1) with a correlation coefficient of 0.9949 (n=9) and a detection limit of 3.8 x 10(-9) mol L(-1) at 3 sigma. The modified electrodes displayed excellent electrochemical response, high sensitivity, long-term stability, and good bioactivity and selectivity.

Synthesis and electrochemical properties of single-walled carbon nanotube–gold nanoparticle composites

We report a facile and environmentally friendly, one-step procedure that allows the aqueous synthesis of a nanocomposite composed of single-walled carbon nanotubes (SWNTs) and gold nanoparticles. In this procedure, chitosan (a nontoxic natural polysaccharide) served as a polymer for wrapping SWNTs, allowing dispersion of SWNTs in aqueous solution, and also functions both as a reducing agent for gold cations and a stabilizing agent for the resulting gold nanoparticles (GNPs). The synthesized GNPs are shown by TEM to coat the side walls of the carbon nanotubes (CNTs). We report UV–vis, Raman, and electrochemical measurements that provide information concerning properties of the resulting nanocomposite. A discussion is provided regarding the use of a film composed of the nanocomposite on a gold electrode to promote the direct electron transfer of immobilized microperoxidase-11 (MP-11). Additionally, our studies indicate that the immobilized MP-11 retains its bioelectrocatalytic activity for the reduction of oxygen.

Soft-Landed Protein Voltammetry: A Tool for Redox Protein Characterization

Microperoxidase-11 (MP-11) was first soft landed onto the gold surface of a screen-printed electrode. Intact protein deposition was verified by time-of-flight secondary ion mass spectrometry. The coupling of soft landing with electrochemical techniques allowed unique information to be obtained about the deposition features. A full characterization of the direct electron-transfer properties was performed by modeling data obtained from cyclic voltammetry experiments; calculated values of kinetic electron-transfer constant, formal redox potential, and reorganization energy allow us to hypothesize the mechanism involved in soft landing immobilization and demonstrate the different conformation of the enzyme deposited from two different charged species. The strong interaction between MP-11 and the gold surface and long-term stability of the functionalized electrode characterizes the peculiar features of this procedure, which enhance its power with respect to the existing immobilization procedure and ensure its suitability for those practical applications that could benefit from an unmediated bridgeless bioeletrochemical electron transfer (e.g., biosensor transducers or electrode elements in biofuel cells).

Boolean Logic Gates that Use Enzymes as Input Signals

Biochemical systems that demonstrate the Boolean logic operations AND, OR, XOR, and InhibA were developed by using soluble compounds, which represent the chemical "devices", and the enzymes glucose oxidase (GOx), glucose dehydrogenase (GDH), alcohol dehydrogenase (AlcDH), and microperoxidase-11 (MP-11), which operated as the input signals that activated the logic gates. The enzymes were used as soluble materials and as immobilized biocatalysts. The studied systems are proposed to be a step towards the construction of "smart" signal-responsive materials with built-in Boolean logic.

Kinetic analysis for suicide-substrate inactivation of microperoxidase-11: A modified model for bisubstrate enzymes in the presence of reversible inhibitors

Kinetics of microperoxidase-11 (MP-11) as a heme–peptide enzyme model in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied in the presence of amino acids, taking into account the inactivation of MP-11 during reaction by its suicide substrate, H 2O 2. Reliability of the kinetic equation was evaluated by non-linear mathematical fitting. Fitting of experimental data into a new integrated kinetic relation showed a close match between the kinetic model and the experimental data. Indeed, it was found that the mechanism of suicide-peroxide inactivation of MP-11 in the presence of amino acids is different from MP-11 and/or horseradish peroxidase. In this mechanism, amino acids compete with hydrogen peroxide for the sixth co-ordination position of iron atom in the heme group through a competitive inhibition mechanism. The proposed model can successfully determine the kinetic parameters including inactivation by hydrogen peroxide as well as the inhibitory rate constants by the amino acid inhibitor. Kinetic parameters of inactivation including the initial activity of MP-11, α 0, the apparent inactivation rate constant, k i and the apparent inhibition rate constant for cysteine, k I were obtained 0.282 ± 0.006 min −1, 0.497 ± 0.013 min −1 and 1.374 ± 0.007 min −1 at [H 2O 2] = 1.0 mM, 27 °C, phosphate buffer 5.0 mM, pH 7.0. Results showed that inactivation and inhibition of microperoxidase as a peroxidase model enzyme occurred simultaneously even at low concentrations of hydrogen peroxide (0.4 mM). This kinetic analysis based on the suicide-substrate inactivation of microperoxidase-11, provides a tool and model for studying peroxidase models in the presence of reversible inhibitors. The introduced inhibition procedure can be used in designing activity tunable and specific protected enzyme models in the hidden and reversibly inhibited forms, which do not undergo inactivation.

Electrochemistry of surface wired redox protein: Axial ligation and control of redox potential

Electrochemical characteristics of surface wired heme protein, microperoxidase-11 (MP-11) and the influence of the axial binding of ligand on the redox potential are described. MP-11 was covalently wired on a carboxylic acid terminated self-assembled monolayer (SAM) preassembled on a polycrystalline Au electrode. Well-defined reversible voltammetric response was obtained at −0.43 V for the surface wired MP-11 in phosphate buffer solution (PBS). The effect of axial ligation on the redox properties of surface wired MP-11 has been investigated using amino acids and various N-donor ligands. These ligands replace water molecule axially coordinated to the heme iron and control the formal potential ( E 0 ′ ) of surface confined MP-11. The axial ligands control the E 0 ′ of MP-11 and the E 0 ′ depends on the nature of axial ligand. Histidine, methionine, and pyridines induce a positive shift in the E 0 ′ whereas tryptophan and imidazole cause negative shift in the E 0 ′ . Positive shift in the redox potential implies that the Fe(II) bind these ligands more firmly than Fe(III). The negative shift indicates that the Fe(III) state is stabilized over Fe(II) state. The heterogeneous electron transfer rate constant for the surface wired MP-11 is significantly enhanced upon axial coordination of histidine and 4- N, N′-dimethylaminopyridine (DMAP). This ligand responsive redox behavior demonstrates that the surface wired MP-11 can be successfully used as redox-receptor for recognizing target ligands. The surface wired redox receptor shows many advantages over the solution based receptor. The axial coordination of exogeneous ligands were further confirmed by spectral measurements in homogeneous solution.

Hemozymes Peroxidase Activity Of Artificial Hemoproteins Constructed From the Streptomyces lividans Xylanase A and Iron(III)-Carboxy-Substituted Porphyrins

To develop artificial hemoproteins that could lead to new selective oxidation biocatalysts, a strategy based on the insertion of various iron-porphyrin cofactors into Xylanase A (Xln10A) was chosen. This protein has a globally positive charge and a wide enough active site to accommodate metalloporphyrins that possess negatively charged substituents such as microperoxidase 8 (MP8), iron(III)-tetra-alpha4-ortho-carboxyphenylporphyrin (Fe(ToCPP)), and iron(III)-tetra-para-carboxyphenylporphyrin (Fe(TpCPP)). Coordination chemistry of the iron atom and molecular modeling studies showed that only Fe(TpCPP) was able to insert deeply into Xln10A, with a KD value of about 0.5 microM. Accordingly, Fe(TpCPP)-Xln10A bound only one imidazole molecule, whereas Fe(TpCPP) free in solution was able to bind two, and the UV-visible spectrum of the Fe(TpCPP)-Xln10A-imidazole complex suggested the binding of an amino acid of the protein on the iron atom, trans to the imidazole. Fe(TpCPP)-Xln10A was found to have peroxidase activity, as it was able to catalyze the oxidation of typical peroxidase cosubstrates such as guaiacol and o-dianisidine by H2O2. With these two cosubstrates, the KM value measured with the Fe(TpCPP)-Xln10A complex was higher than those values observed with free Fe(TpCPP), probably because of the steric hindrance and the increased hydrophobicity caused by the protein around the iron atom of the porphyrin. The peroxidase activity was inhibited by imidazole, and a study of the pH dependence of the oxidation of o-dianisidine suggested that an amino acid with a pKA of around 7.5 was participating in the catalysis. Finally, a very interesting protective effect against oxidative degradation of the porphyrin was provided by the protein.

Multiple Logic Gates Based on Electrically Wired Surface-Reconstituted Enzymes

The electrode functionalized with glucose oxidase (GOx) and microperoxidase-11 (MP-11) performs various Boolean logic operations (OR, XOR, AND-OR) upon addition of glucose and/or H2O2 and application of different potentials. The output signal coming from the electrically wired enzymes is electrochemically readable, allowing interfacing of biochemical computing systems with ordinary electronics.

Elaboration of a new hydrogen peroxide biosensor using microperoxidase 8 (MP8) immobilized on a polypyrrole coated electrode

A novel strategy for elaborating a new biosensor for hydrogen peroxide has been developed by combining the known properties of microperoxidase 8 (MP8) as an oxidation catalyst, and the interesting properties of conducting polypyrrole as a supporting matrix to allow a good bioelectrochemical interface and a large dispersion of MP8 on the modified glassy carbon electrode. MP8 was immobilized into the conducting polypyrrole by entrapment during the electrochemical polymerization, and the modified electrode was characterized both by electrochemical and FT-IR measurements. We demonstrated that MP8 could be immobilized into polypyrrole and could undergo an efficient electron transfer. The obtained modified electrode showed a high catalytic activity toward H 2O 2 without the need for an electron mediator. A linear calibration curve was obtained by amperometric measurement at a potential of − 0.1 V/ECS for concentrations of H 2O 2 ranging from 1 to 10 μM. The detection limit obtained was 1 nM which constituted a real improvement, by about three orders of magnitude, when compared to the values reported for other systems using an electrochemical detection.