Effect of proximal ligand substitutions on the carbene and nitrene transferase activity of myoglobin

Engineered myoglobins were recently shown to be effective catalysts for abiological carbene and nitrene transfer reactions. Here, we investigated the impact of substituting the conserved heme-coordinating histidine residue with both proteinogenic (Cys, Ser, Tyr, Asp) and non-proteinogenic Lewis basic amino acids (3-(3′-pyridyl)-alanine, p-aminophenylalanine, and β-(3-thienyl)-alanine), on the reactivity of this metalloprotein toward these abiotic transformations. These studies showed that mutation of the proximal histidine residue with both natural and non-natural amino acids result in stable myoglobin variants that can function as both carbene and nitrene transferases. In addition, substitution of the proximal histidine with an aspartate residue led to a myoglobin-based catalyst capable of promoting stereoselective olefin cyclopropanation under nonreducing conditions. Overall, these studies demonstrate that proximal ligand substitution provides a promising strategy to tune the reactivity of myoglobin-based carbene and nitrene transfer catalysts and provide a first, proof-of-principle demonstration of the viability of pyridine-, thiophene-, and aniline-based unnatural amino acids for metalloprotein engineering.     

A Hemin-Analogous Corrphycene Derivative: Suppression of Heme Oxygenase and Reconstitution with Apomyoglobin

Summary. A closely hemin-analogous corrphycene derivative was prepared in good overall yield. By spectroscopic measurements it was shown that it complexes with the stress protein heme oxygenase and apomyoglobin in a similar way as hemin. However, due to its molecular structure it is not attacked by heme oxygenase, but is able to block this enzyme to some degree. In addition, the complex with apomyoglobin displays oxygen and carbon monoxide ligation comparable to myoglobin. These properties make this novel corrphycene derivative a candidate to be used as heme oxygenase blocker or otherwise as a blood pigment substitute.Received July 15, 2003; accepted September 5, 2003 Published online October 23, 2003     

Bioelectrochemical dehalogenations via direct electrochemistry of poly(ethylene oxide)-modified myoglobin

Catalytic reductive dehalogenation of hexachloroethane (HCE) was shown to be possible using a graphite electrode coated with a film of the poly(ester sulfonic acid) containing myoglobin (Mb). The effectiveness of the dehalogenation was limited by the solubility of HCE in aqueous solutions. In order to produce an electrode that could dehalogenate HCE in non-aqueous solutions, Mb was chemically modified by addition of poly(ethylene oxide) (PEO). The PEO–Mb–AQ29D modified electrode was found to be suitable for the reductive dehalogenation of HCE in ethanolic solutions. The catalytic response was shown to be linearly dependent on the bulk concentration of HCE.     

Crystal structure and peroxidase activity of myoglobin reconstituted with iron porphycene

The incorporation of an artificially created metal complex into an apomyoglobin is one of the attractive methods in a series of hemoprotein modifications. Single crystals of sperm whale myoglobin reconstituted with 13,16-dicarboxyethyl-2,7-diethyl-3,6,12,17-tetramethylporphycenatoiron(III) were obtained in the imidazole buffer, and the 3D structure with a 2.25-A resolution indicates that the iron porphycene, a structural isomer of hemin, is located in the normal position of the heme pocket. Furthermore, it was found that the reconstituted myoglobin catalyzed the H2O2-dependent oxidations of substrates such as guaiacol, thioanisole, and styrene. At pH 7.0 and 20 degrees C, the initial rate of the guaiacol oxidation is 11-fold faster than that observed for the native myoglobin. Moreover, the stopped-flow analysis of the reaction of the reconstituted protein with H2O2 suggested the formation of two reaction intermediates, compounds II- and III-like species, in the absence of a substrate. It is a rare example that compound III is formed via compound II in myoglobin chemistry. The enhancement of the peroxidase activity and the formation of the stable compound III in myoglobin with iron porphycene mainly arise from the strong coordination of the Fe-His93 bond.     

Electroanalysis of myoglobin and hemoglobin with a boron-doped diamond electrode

The electrochemical behavior of myoglobin (Mb) and hemoglobin (Hb) was investigated with a boron-doped diamond (BDD) electrode by cyclic voltammetry. In acetate buffer solutions, the oxygen reduction at the BDD electrode showed a very high overpotential while the reduction of Mb or Hb was observed in the more positive potential region. Owing to the electrocatalytic reaction of O₂ and the participation of H⁺ following the electrochemical reduction of ferric proteins, the voltammetric responses for Mb and Hb on the BDD electrode in the negative going scans became remarkable in acidic buffer solutions in air. The peak current was linearly proportional to the concentration of Mb in the range 1×10⁻⁶–2×10⁻⁵ M or the concentration of Hb from 1×10⁻⁶ to 1×10⁻⁵ M.     

Chemiluminescence lateral flow immunoassay based on Pt nanoparticle with peroxidase activity

A lateral flow immunoassay (LF-immunoassay) with an enhanced sensitivity and thermostability was developed by using Pt nanoparticles with a peroxidase activity. The Pt nanoparticles were synthesized by citrate reduction method, and the peroxidase activity of Pt nanoparticles was optimized by adjusting reaction conditions. The peroxidase activity was estimated by using Michaelis–Menten kinetics model with TMB as a chromogenic substrate. The kinetics parameters of K_M and V_max were calculated and compared with horseradish peroxidase (HRP). The thermal stability of the Pt nanoparticles was compared with horseradish peroxidase (HRP) according to the storage temperature and long-term storage period. The feasibility of lateral flow immunoassay with a chemiluminescent signal band was demonstrated by the detection of human chorionic gonadotropin (hCG) as a model analyte, and the sensitivity was determined to be improved by as much as 1000-fold compared to the conventional rapid test based on colored gold-colloids.     

Slipping of a histidine improved the peroxidase activity of a de novo designed polypeptide packing an iron porphyrin

Polypeptides with two histidines and an iron porphyrin (1H⁴⁰-7H⁴⁶) were synthesized with a variety of positions of a histidine. In 4H⁴³, histidine (H⁴³) was in the hydrophobic region of an α-helix. The other polypeptides were of slightly or substantially distorted conformation. In the pH 7.2 buffer solution, two histidines of the polypeptide coordinated the iron porphyrin regardless of their positions. Some polypeptides (1H⁴⁰, 3H⁴², and 5H⁴⁴) showed an enhanced catalytic activity in the peroxidase reaction using cumene hydroperoxide compared to that of 4H⁴³, whereas some polypeptides (2H⁴¹ and 6H⁴⁵) were ineffective catalysts. The distortion of the peptide conformation by the addition of MeOH was also effective for the peroxidase reaction.     

Electrochemical behavior of electrodeposited film derived from rhein and its activity towards myoglobin reduction

The stable electroactive thin film of rhein has been investigated by cyclic voltammetry and electrochemical impedance spectroscopy. Electrochemical impedance spectroscopy of the electrodeposited film derived from rhein indicated the electrode reaction was kinetically controlled in the region of higher frequency, the charge transfer resistance was 2.6×10³ Ω cm² and capacitance value was 13.2 μF cm² . The electrodeposited film derived from rhein exhibited a good electrocatalytic activity for myoglobin (Mb) reduction. In 0.30 mol dm⁻³ H₂SO₄solution, the catalysis currents were proportional to the concentrations of Mb over the range of 1.5×10⁻⁷–1.3×10⁻⁵ mol dm⁻³. The detection limit is 1.0×10⁻⁷ mol dm⁻³ (S/N=3). The relative standard deviation is 4.8% for eight successive determinations of 5.0×10⁻⁷ mol dm⁻³ Mb.     

Application of factorial design to the optimization of peroxidase activity in reverse micelles of bis(2-ethylhexyl)sodium sulfosuccinate/isooctane

Two cationic peroxidases isolated from Vaccinium myrtillus were encapsulated in reverse micelles of bis(2-ethylhexyl)sodium sulfosuccinate/isooctane. By using a central composite design, some relevant parameters for the enzymatic activity, such as surfactant and water concentration, pH, and buffer molarity, were analyzed. With the results obtained from this experimental planning, the response surface curves were established. The maximum specific activity obtained (0.19 mM/min · mM of enzyme) was approximately the same for both peroxidases, but the experimental conditions under which this value was attained differed considerably.     

Direct electrochemistry and spectroelectrochemistry of osmium substituted horseradish peroxidase

In this contribution the substitution of the central protoporphyrin IX iron complex of horseradish peroxidase by the respective osmium porphyrin complex is described. The direct electrochemical reduction of the Os containing horseradish peroxidase (OsHRP) was achieved at ITO and modified glassy carbon electrodes and in combination with spectroscopy revealed the three redox couples Os^IIIHRP/Os^IVHRP, Os^IVHRP/Os^VHRP and Os^VHRP/Os^VIHRP. The midpoint potentials differ dependent on the electrode material used with E_1/2 (Os^III/IV) of − 0.4 V (ITO) and − 0.25 V (GC), E_1/2 (Os^IV/^V) of − 0.16 V (ITO) and + 0.10 V (GC), and E_1/2 (Os^V/VI)of + 0.18 V (ITO), respectively. Moreover, with immobilised OsHRP the direct electrocatalytic reduction of hydrogen peroxide and tert-butyl hydroperoxide was observed. In comparison to electrodes modified with native HRP the sensitivity of the OsHRP-electrode for tert-butyl hydroperoxide is higher.     

A study of the chemiluminescence behavior of myoglobin with luminol and its analytical applications

A chemiluminescence signal at 425 nm was observed when ferric state myoglobin was mixed with luminol in alkaline medium. Because the signal was remarkably enhanced in the presence of Fe(CN)₆ ^4–, analytical applications were investigated in a flow-injection system. The increase in chemiluminescence was linearly dependent on myoglobin concentration in the range 0.1 to 100 nmol L^–1, and the limit of detection was 0.04 nmol L^–1 with relative standard deviation 3.2% (3). It was also found that binding of Mb with the ligands CN^–, SCN^–, and F^– significantly inhibited the chemiluminescence reaction. The linear dynamic ranges for the ligands were 1.0–300.0, 0.1–3.0, and 0.5–100.0 nmol L^–1, and the limits of detection (S/N=3) 0.4, 0.04, and 0.2 nmol L^–1, for F^–, CN^–, and SCN^–, respectively. The relative standard deviations were 5.32%, 6.13%, and 3.38% for 0.1 nmol L^–1 CN^–, 0.5 nmol L^–1 SCN^–, and 1.0 nmol L^–1 F^–, respectively. At a flow rate of 2.0 mL min^–1 the assay could be accomplished in 1 min, including sampling and washing. The method has been successfully applied to the determination of myoglobin in human urine and F^– in water samples. A possible mechanism of chemiluminescence production by myoglobin and luminol is presented.     

Improved activity of horseradish peroxidase (HRP) in ‘specifically designed’ ionic liquid

The ionic liquid (IL), tetrakis (2-hydroxyethyl) ammonium triflouromethanesulfonate is rationally designed for horseradish peroxidase (HRP) on the basis of its stability and activity in the presence of an excipient, tris(hydroxymethyl)aminoethane (TRIS) in different ILs. The activity of HRP in this tailor made IL is at least 30-240-fold higher than that in conventional ILs. Also, the activity is more than 10 times greater than that in methanol, a common organic solvent used for HRP.     

Direct detection of myoglobin in whole blood using a disposable amperometric immunosensor

An amperometric immunosensor for the rapid detection of myoglobin in whole blood was developed. Due to its rapid kinetics, myoglobin is a useful biochemical marker for the early assessment of acute myocardial infarction (AMI). A one-step indirect sandwich assay was employed using a polyclonal goat anti-human cardiac myoglobin antibody with monoclonal mouse anti-myoglobin and goat anti-mouse IgG conjugated to alkaline phosphatase (AP), as the detecting antibodies. The final sensor required 30 min for incubation. The standard curve was linear between 85 and 925 ng/ml. The intra- and inter-assay coefficients of variation were below 8%. No cross-reactivity of the antibodies was found with other cardiac proteins. The overall performance of the sensor, rapid analysis time, wide working range, good precision and specificity demonstrate its potential usefulness for early assessment of AMI.     

Fast LC separation of a myoglobin digest: a case study using monolithic and particulate RP 18 silica capillary columns

A method was developed for the fast separation of a myoglobin digest using a monolithic RP 18 silica capillary column of 100 μm I.D. The results were compared with those obtained with a particulate RP 18 silica capillary column of 100 μm I.D. at a flow-rate between 0.6 and 1.2 μl/min. The digest was analyzed at the monolithic column at a flow-rate up to 2.8 μl/min. This high flow-rate could not be applied to the particulate column due to the high back-pressure. When the starting composition of the gradient was changed from 0 to 20% and a gradient steepness of 16%/min was used, the analysis time was less than 4 min. A positive Mascot identification score of 115 was achieved for the MS–MS data. When a lower gradient steepness was employed, the chromatographic resolution and the peak capacity did not increase for most compounds. The intraday repeatability for the retention time of the monolithic column was better than 1.5% at 2.8 μl/min and even less than 0.5% using a flow-rate of 0.6 or 1.0 μl/min. For the particulate column, it was between 0.5 and 1.4% for a flow-rate of 0.6 μl/min, probably due to the high column back-pressure. The interday reproducibility for the retention time of the monolithic column was less than 0.9% using a flow-rate of 1.0 μl/min.     

Layer-by-layer films of hemoglobin or myoglobin assembled with zeolite particles: electrochemistry and electrocatalysis

Positively charged hemoglobin (Hb) or myoglobin (Mb) at pH 5.0 in solutions and negatively charged zeolite particles in dispersions were alternately adsorbed onto solid surfaces forming [zeolite/protein](n) layer-by-layer films, which was confirmed by quartz crystal microbalance (QCM) and cyclic voltammetry (CV). The protein films assembled on pyrolytic graphite (PG) electrodes exhibited a pair of well-defined, nearly reversible CV peaks at about -0.35 V vs. SCE at pH 7.0, characteristic of the heme Fe(III)/Fe(II) redox couples. Hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)) in solution were catalytically reduced at [zeolite/protein](7) film modified electrodes, and could be quantitatively determined by CV and amperometry. The shape and position of infrared amide I and II bands of Hb or Mb in [zeolite/protein](7) films suggest that the proteins retain their near-native structure in the films. The penetration experiments of Fe(CN)(6)(3-) as the electroactive probe into these films and scanning electron microscopy (SEM) results indicate that the films possess a great amount of pores or channels. The porous structure of ]zeolite/protein](n) films is beneficial to counterion transport, which is crucial for protein electrochemistry in films controlled by the charge-hopping mechanism, and is also helpful for the diffusion of catalysis substrates into the films. The proteins with negatively charged net surface charges at pH 9.0 were also successfully assembled with like-charged zeolite particles into layer-by-layer films, although the adsorption amount was less than that assembled at pH 5.0. The possible reasons for this were discussed, and the driving forces were explored.     

Measurement of peroxidase activity in single neutrophils by combining catalyzed-enzyme reaction and epi-fluorescence microscopy

An epi-fluorescence microscopy for determination of peroxidase in individual neutrophils was developed by a combination of enzyme-catalyzed reaction and fluorescence detection. In this method, an individual cell was transferred into a microliter-volume vessel and lysed by freeze-thawing and ultrasonication. The peroxidases-catalyzed reaction was initiated by adding the buffer solution containing nonfluorescent substrates 10-acetyl-3,7-dihydroxyphenoxazine and H₂O₂ to the vessel. Peroxidase activity could be determined via measuring the fluorescent signal of the product resorufin of enzyme-catalyzed reaction. When the slope of kinetic curve of the enzyme-catalyzed reaction was used to quantify peroxidases in single cells, the effect of the time difference between each measurement and the interference from other intracellular compounds that could emit fluorescence can be eliminated.     

In silico locating the immune-reactive segments of Lepidium draba peroxidase and designing a less immune-reactive enzyme derivative

Peroxidases have broad applications in industry, environmental as well as pharmaceutical and diagnosis. Recently applicability of peroxidases in cancer therapy was mentioned. In the present study, a horseradish peroxidase homologue from Lepidium draba was subjected to in silico analyzes aiming at identifying and locating immune-reactive regions. A derivative sequence with decreased immunogenicity and increased stability also suggested. The tertiary structure of the enzyme was predicted. The functional and structural importance of residues was annotated as well as the conservatory status of each residue. The immune-dominant regions of protein were predicted with various software. N-terminal 4 residues, NFSHTGL (186–192), PRNGN (210–214), PLVRAYADGTQKFFN (261–275), and last 4 residues in C-terminal were predicted to be the consensus immunogenic segments of L. draba peroxidase. The modifications were applied to wild type sequence in order to mitigate its immune-reactiveness. The modifications were based on predicted energetic status of residues and naturally occurred amino acids in each position of the enzyme sequence, extracted from alignment file of 150 homologous peroxidases. The new enzyme derivative is predicted to be less immune-reactive and more stable. Thus the sequence is better suited to therapeutic applications.     

Direct electron-transfer of myoglobin within a new zwitterionic gemini surfactant film and its analytical application for H2O2 detection

The direct electron-transfer of myoglobin in a new zwitterionic gemini surfactant film with glassy carbon electrode surface has been investigated. A pair of well-defined and quasi-reversible voltammetric peaks was observed at −0.34 and −0.30 V due to the direct electron-transfer of the redox couple of Mb (Fe^III/Fe^II). The voltammetric responses of myoglobin–surfactant film under different pH and scan rate conditions were obtained. The presence of hydrogen peroxide changed the typical electrochemical behaviors in terms of bioelectrocatalysis of myoglobin to hydrogen peroxide, and a higher sensitive electroanalytical method for the determination of hydrogen peroxide has been developed.