Binding Of Myoglobin Research Articles

Exploring the underlying mechanisms on NaCl-induced reduction in digestibility of myoglobin

In this paper, the structural changes of myoglobin after salt treatment and the binding mode of substrate and pepsin/trypsin were studied by ultraviolet absorption spectroscopy, synchronous fluorescence spectroscopy, LC-MS/MS and molecular dynamics simulation. NaCl treatment changed the heme structure and hydrophobic cavity of myoglobin, which in turn decreased the digestibility of the protein. Trypsin was more effective for digestion of high molecular weight peptides. Molecular dynamics simulation showed that the binding of myoglobin and pepsin/trypsin was mainly maintained by hydrogen bond and hydrophobicity. In addition, the catalytic active center of pepsin is not suitable for long-time existence of low-molecular weight peptide under salt treatment. The findings provide a new insight into the low bioavailability of myoglobin.

Spectral Study of the Interaction of Myoglobin with Tannin

The interaction of myoglobin with tannin (tannic acid) at 298.15 and 303.15 K was studied by fluorescence and absorption spectroscopy in the UV region. The physicochemical and thermodynamic binding parameters (the fluorescence quenching mechanism, the bonding constant, the number of binding sites, the type of interaction) and parameters of the formed complex were determined. It was found that binding of myoglobin with tannic acid does not lead to significant changes in the electronic state of the heme ring of myoglobin.

Critical processing parameters of carbon dioxide spray drying for the production of dried protein formulations: A study with myoglobin

The aim of this study was to gain fundamental insight into protein destabilization induced by supercritical CO2 spray drying processing parameters. Myoglobin was used as a model protein (5mg/ml with 50mg/ml trehalose in 10mM phosphate buffer, pH 6.2). The solution was exposed to sub- and supercritical CO2 conditions (65–130bar and 25–50°C), and CO2 spray drying under those conditions. The heme binding of myoglobin was determined by UV/Vis, fluorescence, and circular dichroism spectroscopy, while myoglobin aggregation was studied by using size-exclusion chromatography and flow imaging microscopy. It was found that pressure and temperature alone did not influence myoglobin’s integrity. However, when pressurized CO2 was introduced into myoglobin solutions at any condition, the pH of the myoglobin formulation shifted to about 5 (measured after depressurization), resulting in heme binding destabilization and aggregation of myoglobin. When exposed to CO2, these degradation processes were enhanced by increasing temperature. Heme binding destabilization and myoglobin aggregation were also seen after CO2 spray drying, and to a greater extent. Moreover, the CO2 spray drying induced the partial loss of heme. In conclusion, pressurized CO2 destabilizes the myoglobin, leading to heme loss and protein aggregation upon spray drying.

Reflectometric interference spectroscopy-based immunosensing using immobilized antibody via His-tagged recombinant protein A

The proposed approach demonstrated in this study provides an immunosensing system based on reflectometric interference spectroscopy (RIfS) in combination with an antibody immobilization method using histidine-tagged recombinant protein A. Carboxymethyldextran (CMD) was immobilized on a 3-aminopropyltriethoxysilane-treated a silicon nitride-coated silicon wafer, followed by chelating histidine-tagged recombinant protein A with copper (II) ions. The CMD-layer was found to be advantageous in terms of not only immobilization of histidine-tagged recombinant protein A-mediated an antibody against myoglobin (anti-Myo) but also prevention of non-specific binding of myoglobin. Myoglobin was repeatedly detected, and the apparent detection limit was 0.1μgmL(-1). The proposed RIfS-based protein sensing system, in conjunction with the easy preparation of silicon-based inexpensive immunosensing chips, is expected to be applicable for label-free optical detection for other proteins in various fields.

Biofunctionalization of α-Zirconium Phosphate Nanosheets: Toward Rational Control of Enzyme Loading, Affinities, Activities and Structure Retention

Controlling the properties of enzymes bound to solid surfaces in a rational manner is a grand challenge. Here we show that preadsorption of cationized bovine serum albumin (cBSA) to α-Zr(IV) phosphate (α-ZrP) nanosheets promotes enzyme binding in a predictable manner, and surprisingly, the enzyme binding is linearly proportional to the number of residues present in the enzyme or its volume, providing a powerful, new predictable tool. The cBSA loaded α-ZrP (denoted as bZrP) was tested for the binding of pepsin, glucose oxidase (GOX), tyrosinase, catalase, myoglobin and laccase where the number of residues increased from the lowest value of ∼153 to the highest value of 2024. Loading depended linearly on the number of residues, rather than enzyme charge or its isoelectric point. No such correlation was seen for the binding of these enzymes to α-ZrP nanosheets without the preadsorption of cBSA, under similar conditions of pH and buffer. Enzyme binding to bZrP was supported by centrifugation studies, powder X-ray diffraction and scanning electron microscopy/energy-dispersive X-ray spectroscopy. All the bound enzymes retained their secondary structure and the extent of structure retention depended directly on the amount of cBSA preadsorbed on α-ZrP, prior to enzyme loading. Except for tyrosinase, all enzyme/bZrP biocatalysts retained their enzymatic activities nearly 90-100%, and biofunctionalization enhanced the loading, improved structure retention and supported higher enzymatic activities. This approach of using a chemically modified protein to serve as a glue, with a predictable affinity/loading of the enzymes, could be useful to rationally control enzyme binding for applications in advanced biocatalysis and biomedical applications.

Binding of hemoglobin to ultrafine carbon nanoparticles: a spectroscopic insight into a major health hazard

Binding of hemoglobin and myoglobin to carbon nanoparticles.

Effect of Ionic Strength and Temperature on Interaction between Fish Myoglobin and Myofibrillar Proteins

The effects of ionic strength (0, 0.3, and 0.6 M KCl) and temperature (4 and 25 degrees C) on the interaction between fish myoglobin and myofibrillar proteins were investigated in a model system. Increases in the relative content of bound myoglobin and metmyoglobin formation in myoglobin-natural actomyosin (NAM) mixtures with concurrent decreases in whiteness and Ca(2+)-ATPase activity were observed with increasing ionic strength (P < 0.05). The relative content of bound myoglobin and the oxidation of oxymyoglobin were generally greater at 25 degrees C than at 4 degrees C (P < 0.05). Binding of myoglobin to NAM resulted in decreased whiteness (P < 0.05). Ca(2+)-ATPase was not affected by temperature (P > 0.05). SDS-PAGE patterns of protein samples suggested that myoglobin-NAM interactions did not involve disulfide bonds. The formation of high-molecular-weight aggregates (>206 kDa) was observed and was more pronounced at higher ionic strength and higher temperature.

Determination of picomole amounts of formaldehyde in air and bio-fluids based on its enhanced myoglobin-luminol chemiluminescence reaction

A rapid and sensitive chemiluminescence determination of formaldehyde with the flow injection technique was presented. It was found that the specific binding of myoglobin with formaldehyde could accelerate the electron transfer between myoglobin and luminol, thus producing quicker and more efficient chemiluminescence at 425 nm than with the myoglobin–luminol reaction. The increased chemiluminescence intensity was linear with the formaldehyde concentration in the range of 1–3000 pmol L−1, and the limit of detection was 0.3 pmol L−1 with a relative standard deviation of less than 5.0% (3σ). At a flow rate of 2.0 mL min−1, including sampling and washing, the assay could be accomplished in 0.5 min. It was also found that there was a linear relationship between the vapour pressure of formaldehyde and temperatures of 291.5–315.0 K. The proposed method has been applied successfully for the determination of formaldehyde in air and human serum.

Utilization of myoglobin as a heme source byHaemophilus influenzaerequires binding of myoglobin to haptoglobin

Haemophilus influenzae has an absolute growth requirement for heme. One potential in vivo source of heme is the protein myoglobin which is found at low levels in human serum. No tested H. influenzae strain was able to use myoglobin as a heme source. However, all strains were able to utilize the heme from myoglobin when myoglobin was complexed with haptoglobin. Utilization of the haptoglobin-myoglobin complex was shown to be mediated by the previously described hemoglobin/hemoglobin-haptoglobin-binding proteins of H. influenzae.

In Situ Investigation of Coadsorption of Myoglobin and Methylene Blue to Hydrophilic Glass by Broadband Time-Resolved Optical Waveguide Spectroscopy

Recently, we have developed a broadband optical waveguide (OWG) spectrometer by using commercially available glass plates of tens of micrometers in thickness as the substrate-free multimode waveguides (Qi et al. Opt. Lett. 2002, 27, 2001-2003). The spectrometer having a bandwidth from 360 to 800 nm is capable of simultaneously detecting the Soret-band absorption of heme proteins and the visible absorption of organic dyes. In this article, the spectrometer was used to in situ investigate coadsorption of methylene blue (MB) and myoglobin from the mixed aqueous solution onto bare glass. Both MB and myoglobin in the mixed solution are positively charged, which makes them not only avoid the chemical interaction between each other but also easy to adsorb to hydrophilic glass. It was found that the coadsorption of MB and myoglobin occurred just in the early stage and the glass surface was finally occupied by myoglobin. The OWG spectroscopic investigation into the respective MB and myoglobin adsorptions shows that MB adsorption is reversible to some degree but that of myoglobin is irreversible. It reveals that the electrostatic binding of myoglobin to bare glass is stronger than the case of MB. Therefore, the adsorbed MB can be substituted by myoglobin. Moreover, via the electrostatic repulsion the tightly immobilized myoglobin prevents bulk MB from occupying the empty surface sites. It is the reason MB is absent from the hydrophilic glass coated with a submonolayer of myoglobin. In the article, we explained both the strong dimerization of MB at the interface and a slow decrease with time of the Soret-band absorbance after its maximum was reached. We also estimated the myoglobin coverage based on the waveguide theory. The study shows the distinguished applicability of the broadband OWG spectroscopy for in situ, real-time monitoring of the dye-protein coadsorption to silica from the mixed solution.

Binding and utilization of myoglobin by Porphyromonas gingivalis

Myoglobin was found to bind reversibly to the envelope of Porphyromonas gingivalis in a pH-dependent manner; the binding took place below neutral pHs of the incubation mixtures and myoglobin bound released from the envelope at high pHs. The amounts of myoglobin bound to 1 mg of the envelope at pH 5.0 per min under the presence of sufficient myoglobin were 1.4 μg. K d for the reaction at pH 5.0 was 2.2×10 −10 M. From the dot blot assay, myoglobin obviously bound to hemoglobin-binding protein (HbBP) of P. gingivalis, however, the amounts of myoglobin that bound to HbBP were half those of hemoglobin. One of the fractions, separated by gel filtration, of the digested materials of myoglobin by the detergent-solubilized envelope containing proteinases was found to support the growth of P. gingivalis in the iron source-depleted medium.

Steric Considerations in the Covalent Binding of Myoglobin to Thin Films

Myoglobin is shown to bind to mixed thin films of docosyl mesylate and methyl docosanoate. Myoglobin can be attached to films containing docosyl mesylate; however, films containing only methyl docosanoate exhibit only minimal myoglobin attachment. This indicates that myoglobin is specifically attached to the docosyl mesylate sites on the film surface. Surface coverage of myoglobin is dependent on the percentage of docosyl mesylate in the film. A maximum myoglobin coverage of approximately 1.2 × 1012 myoglobin/cm2 is estimated for films composed of between 22% and 30% docosyl mesylate.

Electrostatic Modulation of Ligand Binding and Electrochemical Properties of Myoglobin: The Role of Charge Compensation

The effect of electrostatic substitutions in the heme pocket of myoglobin on the ligand binding and electrochemical properties of three variants of horse heart myoglobin (S92D, V67R, and V68H) have...

Spectroelectrochemical and electrochemical determination of ligand binding and electron transfer properties of myoglobin, cyanomyoglobin, and imidazolemyoglobin

Abstract Electrochemistry and spectroelectrochemistry were used to study solutions of horse myoglobin, both by itself and complexed with cyanide or imidazole, at indium-tin oxide electrodes. In the absence of ligands other than water, myoglobin exhibited slow quasi-reversible heterogeneous electron transfer kinetics, with a formal heterogeneous electron transfer rate constant ( k °′) value of 1.6(±0.4)×10 −5 cm s −1 . Differences from simple simulations in the reverse sweep (anodic) cyclic voltammograms are proposed to be due to residual amounts of dioxygen in solution and/or in the protein molecule. Those differences were not seen in the anodic waveforms of simultaneously acquired derivative cyclic voltabsorptometry (DCVA) experiments. Cyanomyoglobin exhibited faster heterogeneous electron transfer kinetics, with a k °′ value of 6.0(±1.0)×10 −4 cm s −1 . Values for the homogeneous rate constants for dissociation ( k f ) and association ( k b ) of cyanide from and to the electrochemically reduced protein were determined to be 0.08(±0.03) s −1 and 0.06(±0.03) M −1 s −1 , respectively. Imidazolemyoglobin was found to transfer electrons faster than cyanomyoglobin, with a k °′ value of 2.0(±0.5)×10 −3 cm s −1 . The values for the homogeneous rate constants k f and k b were 0.40(±0.1 5) s −1 and 1.1(±0.1) M −1 s −1 , respectively.

Kinetics of Conformation Change in Cyanometmyoglobin Following Electron Transfer Monitored by Magnetically Induced Circular Dichroism Spectrometry

The characterization of the dynamics of conformational changes that accompany the ligand binding and dissociation reactions of myoglobin may provide insights into the events that control the physiological function of this oxygen storage protein. The cyanometmyoglobin system was chosen for this study because cyanide binds to the metmyoglobin state and dissociates upon reduction. The potential step spectroelectrochemical method was used here because the rate of cyanometmyoglobin reduction at an electrode can be controlled so that the rate of dissociation of the reduced cyanomyoglobin can then be followed.

An Assay for Human Myoglobin Using the Technique of Membrane Immunoassay with Light-Sensitive Nylon Supports

Abstract A method for rapid determination of human myoglobin was developed. The method is based on the technique of membrane immunoassay with light-sensitive nylon membranes for covalent immobilization of antibodies. The kinetic of binding of myoglobin with immobilized antibodies was studied. The pumping of reagents through the pores of a membrane allows to reduce the total assay time to 40 min. The sensitivity of membrane assay of myoglobin was 20 ng/ml. A semi-quantitative method with visual detection also was proposed. It allows to distinguish various states of myocardial infarction pathology.

Consequence of rapid heme rotation to the oxygen binding of myoglobin

The myoglobin complexed with octamethylhemin was prepared. The visible absorption profiles are typical of general alkylhemin-substituted myoglobins, suggesting normal insertion of the hemin into the hydrophobic cavity. The NMR spectra of various ferric proteins were anomalous, without clearly resolved signals from the prosthetic group, most probably reflecting rapid heme rotation about the iron-histidine bond. The equilibrium and kinetic oxygen bindings were measured to examine the functional significance of the heme motion. Functional comparison with the myoglobin having immobile hemin indicates that rotation of the octamethylheme negligibly affects the myoglobin function. The results suggest that neither the heme peripheral contacts nor the proximal imidazole orientation about the heme normal is the dominant factor to control myoglobin function.

Acidification and disulfide reduction can be sufficient to allow intact proteins to bind class II MHC.

The hypothesis that reduction of disulfide bonds and exposure to low pH may be sufficient to allow intact proteins to bind class II MHC was addressed in this study. Functional assays were used to determine minimal conditions sufficient to bypass the requirement for Ag processing. Fixed APC, pulsed with HEL at pH 5 in the presence of a reducing agent, were observed to stimulate I-Ed-restricted T cell hybridomas. Activity required both low pH and reducing agent. Fixed APC also stimulated cytochrome c-specific T cells after exposure to Ag at pH 5 but not pH 7. No reducing agent was required. Peptide was considerably more potent in these assays than intact cytochrome c. By contrast, highly purified cytochrome c was equal in potency with a peptide in competition binding assays using purified I-Ek. The protein did not bind I-Ad or I-Ak. This suggested that cytochrome c can efficiently bind I-Ek at low pH without the requirement for proteolytic cleavage. The capacity of HEL to bind purified I-Ed was strictly dependent on the presence of reducing agent. The reducing agent alone had no effect. A small panel of proteins was screened for class II binding at low pH in competition assays. Horse myoglobin and human hemoglobin were particularly potent in their capacity to inhibit binding of biotin-labeled peptide to purified class II. Unlabeled peptide inhibited binding of labeled peptide to I-Ad at pH 5 and pH 7. By contrast, intact horse myoglobin was active only at pH 5. This result suggested that low pH may enhance the binding of horse myoglobin to I-Ad through effects on the structure of both proteins. Our observations support the hypothesis that low pH and disulfide reduction can be sufficient to allow partially unfolded or structurally destabilized proteins to bind class II MHC.

Measurement of myoglobin diffusivity in the myoplasm of frog skeletal muscle fibres.

1. Experiments were carried out on intact, single skeletal muscle fibres from frog in order to estimate the apparent diffusion constant of myoglobin (denoted DAPP) in the myoplasm of living muscle cells. An optical technique was employed to measure myoglobin concentration along the fibre axis following injection of metmyoglobin (denoted metMb) at a point source. The concentration profiles were fitted by the one-dimensional diffusion equation to give estimates of DAPP. The method relied on the fact that myoglobin is normally absent from these frog fibres, thus permitting resolution of the myoglobin-related absorbance above the intrinsic absorbance of the fibre. 2. One complication in the method was that metMb became significantly reduced to oxymyoglobin (denoted MbO2) during the elapsed time before measurement of the concentration profile. The rate of reduction was evaluated by fitting myoglobin-related absorbance spectra, measured at different times following injection of metMb, with in vitro absorbance spectra of metMb and MbO2. Results from four experiments indicated that reduction could be described by a first-order, irreversible reaction having an average rate constant of 0.0164 min-1 (22 degrees C). The effect of reduction on the fitting of DAPP was taken into account. 3. DAPP was determined under three fibre conditions: (1) long sarcomere spacing (3.6-3.8 microns) at 16 degrees C, (2) long sarcomere spacing at 22 degrees C, and (3) normal sarcomere spacing (2.4-2.7 microns) at 22 degrees C. The average values for DAPP under these conditions were: (1) 0.12 (n = 5); (2) 0.17 (n = 5); and (3) 0.15 (n = 7) x 10(-6) cm2 s-1. The average value at 22 degrees C, 0.16 x 10(-6) cm2 s-1, is about 4 times smaller than values for myoglobin diffusivity at 20 degrees C commonly assumed in models of facilitated transport of oxygen by myoglobin. 4. In order to test the possibility that the unexpectedly low value of DAPP found in intact fibres might be due to the binding of myoglobin to relatively immobile sites in myoplasm, experiments were carried out in a cut-fibre preparation using a technique described by Maylie, Irving, Sizto & Chandler (1987 b) for determining the diffusion constants and degree of myoplasmic binding of absorbance dyes. Values for DAPP and the factor (denoted 1 + beta) by which the total myoglobin concentration exceeded the free myoglobin concentration were obtained by fitting the absorbance data by solutions of the one-dimensional diffusion equation.(ABSTRACT TRUNCATED AT 400 WORDS)

Mapping the idiotopes of a monoclonal anti-myoglobin antibody with syngeneic monoclonal anti-idiotypic antibodies: detection of a common idiotope.

A panel of syngeneic monoclonal anti-idiotypic antibodies was prepared by immunizing A.SW mice with keyhole limpet hemocyanin-coupled A.SW monoclonal anti-myoglobin (HAL 19, IgG1) and screening the cloned hybridomas for production of IgG2 binding to idiotype but not to certain other anti-myoglobin antibodies of the same subclass in an ELISA. With these antibodies, we identified three nonoverlapping idiotopes, based on three clusters of monoclonal anti-idiotopes that mutually inhibit within each cluster, but not between clusters (Cluster I: S2, S6, S8; Cluster II: S5, S7; Cluster III: S9). Only Cluster II antibodies block the binding of myoglobin to HAL 19 and so identify a binding site-related idiotope(s). Binding of both Cluster II monoclonals (S5 and S7) to Hal 19 is inhibited by a rabbit anti-idiotype that we previously reported detects a common cross-reactive anti-myoglobin idiotope in immune sera. However, only one of these, S7, and not S5, identifies an idiotope that is present on 20 to 30% of A.SW anti-myoglobin antibodies in immune sera and ascites. The panel of syngeneic monoclonal anti-idiotype antibodies also detects new idiotopes not detected by the rabbit anti-idiotype. The development of a panel of syngeneic monoclonal anti-idiotypic antibodies to different clusters of idiotopes on the same antibody molecule, including one that identifies a major common idiotope in immune sera, should allow the analysis of possible idiotype network regulation in vivo and in vitro in a completely syngeneic system.