Horse Myoglobin Research Articles

Efficient Electrocatalytic H2 Production by Immobilized Co(III)‐myoglobin

AbstractThe thermodynamics and kinetics of heterogeneous electron transfer (ET) for Co‐substituted horse myoglobin (Co−Mb) and its derivatives with ammonia and imidazole as heme axial ligands were studied with cyclic voltammetry on a pyrolytic graphite electrode along with their ability to mediate the electro‐catalytic production of H2. All the proteins experience a non‐diffusive electrochemical regime as electrode‐bound species. The adsorbed Co−Mb construct carries out the electro‐catalytic reduction of water protons to H2 with a good efficiency under anaerobic conditions thus yielding a simple and tunable system for H2 production. Replacement of H2O as Co axial ligand by ammonia and imidazole significantly lowers the catalytic currents for H3O+/H2O reduction to H2. The E°’ values of the Co(III)/Co(II) redox couple for all species are mainly determined by the enthalpic contribution. Differences were found in the kinetics of ET for the different protein adducts due to changes in the activation enthalpies. However, all species share the same distance of about 14 Å from the electrode surface to the Co(III)/Co(II) center determined using the Marcus model, consistent with a non‐denaturing adsorption of the protein.

Heme Pocket Structure and Its Functional Implications in an Ancestral Globin Protein.

Proteins have undergone evolutionary processes to achieve optimal stability, increased functionality, and novel functions. Comparative analysis of existent and ancestral proteins provides insights into the factors that influence protein stability and function. Ancestral sequence reconstruction allows us to deduce the amino acid sequences of ancestral proteins. Here, we present the structural and functional characteristics of an ancestral protein, AncMH, reconstructed to be the last common ancestor of hemoglobins and myoglobins. Our findings reveal that AncMH harbors heme and that the heme binds oxygen. Furthermore, we demonstrate that the ferrous heme in AncMH is pentacoordinated, similar to that of human adult hemoglobin and horse myoglobin. A detailed comparison of the heme pocket structure indicates that the heme pocket in AncMH is more similar to that of hemoglobin than that of myoglobin. However, the autoxidation of AncMH is faster than that of both hemoglobin and myoglobin. Collectively, our results suggest that ancestral proteins of hemoglobins and myoglobins evolved in steps, including the hexa- to pentacoordination transition, followed by stabilization of the oxygen-bound form.

Oxygen-mediated oxidation of ferrous nitrosylated nitrobindins

The O2-mediated oxidation of all-β-barrel ferrous nitrosylated nitrobindin from Arabidopsis thaliana (At-Nb(II)-NO), Mycobacterium tuberculosis (Mt-Nb(II)-NO), and Homo sapiens (Hs-Nb(II)-NO) to ferric derivative (At-Nb(III), Mt-Nb(III), and Hs-Nb(III), respectively) has been investigated at pH 7.0 and 20.0 °C. Unlike ferrous nitrosylated horse myoglobin, human serum heme-albumin and human hemoglobin, the process in Nb(II)-NO is mono-exponential and linearly dependent on the O2 concentration, displaying a bimolecular behavior, characterized by kon = (6.3 ± 0.8) × 103 M−1 s−1, (1.4 ± 0.2) × 103 M−1 s−1, and (3.9 ± 0.5) × 103 M−1 s−1 for At-Nb(II)-NO, Mt-Nb(II)-NO, and Hs-Nb(II)-NO, respectively. No intermediate is detected, indicating that the O2 reaction with Nb(II)-NO is the rate-limiting step and that the subsequent conversion of the heme-Fe(III)-N(O)OO− species (i.e., N-bound peroxynitrite to heme-Fe(III)) to heme-Fe(III) and NO3− is much faster. A similar mechanism can be invoked for ferrous nitrosylated human neuroglobin and rabbit hemopexin, in which the heme-Fe(III)-N(O)OO− species is formed as well, although the rate-limiting step seems represented by the reshaping of the six-coordinated heme-Fe(III) complex. Although At-Nb(II)-NO and Mt-Nb(II)-NO are partially (while Hs-Nb(II)-NO is almost completely) penta-coordinated, density functional theory (DFT) calculations rule out that the cleavage of the proximal heme-Fe-His bond in Nb(II)-NO is responsible for the more stable heme-Fe(III)-N(O)OO− species. Moreover, the oxidation of the penta-coordinated heme-Fe(II)-NO adduct does not depend on O2 binding at the proximal side of the metal center. These features may instead reflect the peculiarity of Nb folding and of the heme environment, with a reduced steric constraint for the formation of the heme-Fe(III)-N(O)OO− complex.

The Intercalation of CORM-2 with Pharmaceutical Clay Montmorillonite (MMT) Aids for Therapeutic Carbon Monoxide Release.

The pharmaceutical clay montmorillonite (MMT) is, for the first time, explored as a carbon monoxide-releasing material (CORMat). MMT consists of silicate double layered structure; its exfoliation feature intercalate the CORM-2 [RuCl(μ-Cl)(CO)3]2 inside the layers to suppress the toxicity of organometallic segment. The infrared spectroscopy (IR) confirmed the existence of ruthenium coordinated carbonyl ligand in MMT layers. The energy-dispersive X-ray spectroscopy (EDX) analysis showed that ruthenium element in this material was about 5%. The scanning electron microscopy (SEM) and transmission electron microscope (TEM) images showed that the layer-structure of MMT has been maintained after loading the ruthenium carbonyl segment. Moreover, the layers have been stretched out, which was confirmed by X-ray diffraction (XRD) analysis. Thermogravimetric (TG) curves with huge weight loss around 100–200 °C were attributed to the CO hot-release of ruthenium carbonyl as well as the loss of the adsorbed solvent molecules and the water molecules between the layers. The CO-liberating properties have been assessed through myoglobin assay. The horse myoglobin test showed that the material could be hydrolyzed to slowly release carbon monoxide in physiological environments. The half-life of CO release was much longer than that of CORM-3, and it has an excellent environmental tolerance and slow release effect.

Online porous graphic carbon chromatography coupled with tandem mass spectrometry for post‐translational modification analysis

Porous graphic carbon chromatography (PGC) has a different mechanism in the retention of tryptic peptides compared with reversed-phase chromatography and in this study we show that coupling PGC with tandem mass spectrometry offer advantages for the quantitation of phosphorylation stoichiometry and characterization of site-specific glycosylation. Digests of protein standards (horse myoglobin, bovine fetuin and β-casein) were analyzed with a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) system by coupling an Agilent 1100 HPLC system to a Synapt G2-Si HDMS (Waters). Peptides were separated using a HyperCarb PGC column (300 μm i.d. × 100 mm) packed with 3 μm particles. MS/MS data were collected in data-dependent mode and three MS/MS scans were acquired after the full MS scan. RAW data were transformed to .mgf by PLGS (Waters) and searched against the Swissprot database by Mascot. Chromatograms and MS/MS spectra of identified compounds were extracted with Masslynx (Waters) and imported to Origin for analysis. Glycan composition and peptide sequence were manually annotated. PGC/MS/MS enabled accurate quantitation of the stoichiometry of specific phosphorylation sites from β-casein by efficient separation of the phosphopeptide and its non-phosphorylated counterpart, which cannot be achieved by reversed-phase chromatography. PGC/MS/MS also enabled comprehensive characterization of protein sialoglycosylation as isomeric glycopeptides with different combinations of α2-3- and α2-6-linked sialic acids can be separated and the ratios of each combination were verified by exoglycosidase digestion. PGC has demonstrated superior separation of peptides with phosphorylation and glycosylation and can be used as an alternative in the proteomic characterization of post-translational modifications (PTMs) by polar groups.

Physicochemical and conformational studies on interaction of myoglobin with an amino-acid based anionic surfactant, sodium N-dodecanoyl sarcosinate (SDDS)

Physicochemical and conformational studies on the interaction between horse myoglobin, a monomeric water soluble heme protein and sodium N-dodecanoyl sarcosinate (SDDS), an amino-acid based anionic surfactant have been performed at different pHs using surface tension, viscosity, UV–vis and fluorescence spectroscopy, circular dichroism, isothermal titration calorimetry and differential scanning calorimetry. Myoglobin shows surface activity, according to the surface tension measurement. Protein forms aggregates with surfactant molecules and becomes unfolded with increasing SDDS concentration. Again, when concentration of myoglobin increases, the binding ratio decreases; whereas heme changes its spin and coordination number with increasing surfactant concentration. α-Helicity of myoglobin decreases with increase in concentration of surfactant, while β-sheet and random coil appear. The temperature of transition of myoglobin and the enthalpy of thermal denaturation also change at higher concentration of SDDS. The myoglobin-SDDS interaction at different pH, ranging from 5 to 8 has been studied and it shows that pH has very little effect on the conformational change of the myoglobin-SDDS system.

Quantification of species-specific meat proteins in cooked and smoked sausages using infusion mass spectrometry

Label-free quantification combined with high-resolution infusion-based mass spectrometry (MS) was evaluated to authenticate ‘horse sausages’ made from horse meat and pork. Four types of industrially processed sausages, including cooked horse meat, pork and beef, and their mixtures were analysed. Quantitation and evaluation of the species composition were based on a set of 11 species-specific meat proteins and 14 unique heat-stable peptide markers. Using infusion MS, the highest distinguishing value was found in four proteins, namely, horse myosin-7 (MYH7_HORSE) and horse myoglobin (MYG_HORSE), porcine myosin-4 (MYH4_PIG) and bovine myoglobin (MYG_BOVIN). The limit of detection was 5% (w/w) for pork and beef in the three-component matrix and 1% (w/w) for horse meat. The proteins’ abundance was computed using a peak intensity measurement technique for precursor ions, based on the extracted ion currents/intensities of precursor ions. The procedure enabled discrimination between horse meat, pork and beef proteins, as well as estimation of the relative changes in protein abundance in all the examined samples. Substantial differences in the abundance of specific proteins were obtained from the pure meat samples, three-component mixtures and commercial sausages. The method may be useful in the preliminary screening of protein-rich food samples, aimed at fraud detection and estimation of the overall level of adulteration.

Steady-State Kinetics of Phytoglobin-Catalyzed Reduction of Hydroxylamine to Ammonium.

Phytoglobins are plant hexacoordinate hemoglobins with reversible coordination of a histidine side chain to the ligand binding site of the heme iron. They mediate electron transfer reactions such as nitric oxide scavenging and are particularly efficient at reducing nitrite and hydroxylamine. Previous work with phytoglobins has focused only on single turnovers of these reactions and has not revealed whether structural features, such as histidine hexacoordination, play a prominent role in the complete catalytic cycle. This work characterizes steady-state phytoglobin catalysis of reduction of hydroxylamine to ammonium using two different chemical reductants. Km and kcat values were measured for rice phytoglobin, horse myoglobin, human neuroglobin, and a rice phytoglobin mutant protein in which the hexacoordinating histidine has been replaced with leucine (Phyt:H73L). The results demonstrate that phytoglobin catalysis driven by benzyl viologen is limited only by the dissociation rate constant for the distal histidine. This is consistent with the rate limit in single-turnover experiments and demonstrates that the kinetics of hydroxylamine binding, and not phytoglobin reduction, ultimately governs the reaction. Catalysis by the other proteins that either lack or have tighter hexacoordination is much slower, suggesting that facile reversibility of the bond between the distal histidine and the heme iron is needed to allow both substrate binding and heme iron reduction. On the other hand, catalysis driven by dithionite is limited by SO2•- concentrations and is similar for all of these proteins, suggesting that dithionite is not a good reducing agent for evaluation of the catalytic properties of hemoglobins.

Measurement of protein size in concentrated solutions by small angle X-ray scattering.

By simulations on the distance distribution function (DDF) derived from small angle X-ray scattering (SAXS) theoretical data of a dense monodisperse system, we found a quantitative mathematical correlation between the apparent size of a spherically symmetric (or nearly spherically symmetric) homogenous particle and the concentration of the solution. SAXS experiments on protein solutions of human hemoglobin and horse myoglobin validated the correlation. This gives a new method to determine, from the SAXS DDF, the size of spherically symmetric (or nearly spherically symmetric) particles of a dense monodisperse system, specifically for protein solutions with interference effects.

Rapid Myoglobin Aggregation through Glucosamine-Induced α-Dicarbonyl Formation.

The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with N-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a β-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a β-sheet structure.

Avidity of polyreactive immunoglobulins

An analysis of the mechanism of interaction between polyreactive immunoglobulins (PRIG) and antigen was conducted and it was shown that most of the traditional methods of antibody affinity evaluation are not applicable for PRIG affinity. The comparative assessment of the mouse and human PRIG avidity against ovalbumin and horse myoglobin and the avidity of specific monoclonal antibodies against ovalbumin have shown that the avidity of PRIG not only is much less than the avidity of monoclonal antibodies but even exceeds it.

Rational design of heterodimeric protein using domain swapping for myoglobin.

Protein design is a useful method to create novel artificial proteins. A rational approach to design a heterodimeric protein using domain swapping for horse myoglobin (Mb) was developed. As confirmed by X-ray crystallographic analysis, a heterodimeric Mb with two different active sites was produced efficiently from two surface mutants of Mb, in which the charges of two amino acids involved in the dimer salt bridges were reversed in each mutant individually, with the active site of one mutant modified. This study shows that the method of constructing heterodimeric Mb with domain swapping is useful for designing artificial multiheme proteins.

Rational Design of Heterodimeric Protein using Domain Swapping for Myoglobin

AbstractProtein design is a useful method to create novel artificial proteins. A rational approach to design a heterodimeric protein using domain swapping for horse myoglobin (Mb) was developed. As confirmed by X‐ray crystallographic analysis, a heterodimeric Mb with two different active sites was produced efficiently from two surface mutants of Mb, in which the charges of two amino acids involved in the dimer salt bridges were reversed in each mutant individually, with the active site of one mutant modified. This study shows that the method of constructing heterodimeric Mb with domain swapping is useful for designing artificial multiheme proteins.

An improved UPLC method for the detection of undeclared horse meat addition by using myoglobin as molecular marker

In 2013, following the scandal of the presence of undeclared horse meat in various processed beef products across the Europe, several researches have been undertaken for the safety of consumer health. In this framework, an improved UPLC separation method has been developed to detect the presence of horse myoglobin in raw meat samples. The separation of both horse and beef myoglobins was achieved in only seven minutes. The methodology was improved by preparing mixtures with different composition percentages of horse and beef meat. By using myoglobin as marker, low amounts (0.50mg/0.50g, w/w; ∼0.1%) of horse meat can be detected and quantified in minced raw meat samples with high reproducibility and sensitivity, thus offering a valid alternative to conventional PCR techniques.

Concentration-controlled formation of myoglobin/gold nanosphere aggregates.

Gold nanoparticles are being increasingly proposed as biotechnological tools for medical diagnosis and therapy purposes. Their safety for human beings and the environment is therefore becoming an emerging issue, which calls for basic research on the interactions between nanostructured gold particles and biological materials, including physicochemical studies of model systems. In this Article, we focus on the "reaction products" of a widely known nanoparticle type, citrate-capped 30 nm gold nanospheres, with a model protein, horse myoglobin. Protein adsorption and partial denaturation were accompanied by the formation of nanoparticle aggregates with strongly distinct optical spectroscopy properties and shapes, as observed by transmission electron microscopy. We singled out the concentration of myoglobin as the determinant of these differences, and verified on this basis that surface-enhanced Raman scattering (SERS) spectra can only be obtained by aggregates with strong interparticle optical coupling, which are obtained at low protein concentration. The results can be useful both in improving the spectroscopy of biomolecules and in understanding the formation of the protein corona in biomedical applications.

Factors correlating with significant differences between X-ray structures of myoglobin.

Validation of general ideas about the origins of conformational differences in proteins is critical in order to arrive at meaningful functional insights. Here, principal component analysis (PCA) and distance difference matrices are used to validate some such ideas about the conformational differences between 291 myoglobin structures from sperm whale, horse and pig. Almost all of the horse and pig structures form compact PCA clusters with only minor coordinate differences and outliers that are easily explained. The 222 whale structures form a few dense clusters with multiple outliers. A few whale outliers with a prominent distortion of the GH loop are very similar to the cluster of horse structures, which all have a similar GH-loop distortion apparently owing to intermolecular crystal lattice hydrogen bonds to the GH loop from residues near the distal histidine His64. The variations of the GH-loop coordinates in the whale structures are likely to be owing to the observed alternative intermolecular crystal lattice bond, with the change to the GH loop distorting bonds correlated with the binding of specific `unusual' ligands. Such an alternative intermolecular bond is not observed in horse myoglobins, obliterating any correlation with the ligands. Intermolecular bonds do not usually cause significant coordinate differences and cannot be validated as their universal cause. Most of the native-like whale myoglobin structure outliers can be correlated with a few specific factors. However, these factors do not always lead to coordinate differences beyond the previously determined uncertainty thresholds. The binding of unusual ligands by myoglobin, leading to crystal-induced distortions, suggests that some of the conformational differences between the apo and holo structures might not be `functionally important' but rather artifacts caused by the binding of `unusual' substrate analogs. The causes of P6 symmetry in myoglobin crystals and the relationship between crystal and solution structures are also discussed.

Electrochemical determination of heme-linked pKa values and the importance of using fluoride binding in heme proteins

The ultraviolet–visible (UV–vis) spectroelectrochemical measurements of heme proteins in the presence of a heme-bound fluoride ion can be used as a probe for heme-linked ionizations of acid–base groups in the heme pocket. A detailed study of the pH dependence of the midpoint potential of skeletal horse myoglobin (Mb) with a heme-bound fluoride ion (Mb–F) reveals how protonation of the distal histidine (H64) changes the redox properties of the protein with a determined pKa of 5.3. In addition, fluoride binding in myoglobin provides a stabilization of −1.9kcal/mol of the ferric Mb–F relative to ferric Mb without fluoride.

Temperature- and pH-dependent effect of lactate on in vitro redox stability of red meat myoglobins

Our objective was to evaluate the influence of lactate on in vitro redox stability and thermostability of beef, horse, pork, and sheep myoglobins. Lactate (200mM) had no effect (P>0.05) on redox stability at physiological (pH7.4, 37°C) and meat (pH5.6, 4°C) conditions. However, lactate increased (P<0.05) metmyoglobin formation at a condition simulating stressed live skeletal muscle (pH6.5, 37°C). The redox stability of myoglobins at stressed live skeletal muscle and meat conditions was species–specific (P<0.05). Myoglobin thermostability at 71°C was lower (P<0.05) in the presence of lactate compared with controls and was influenced (P<0.05) by species. The results of the present study indicate that the effects of lactate on myoglobin are temperature and pH dependent. The observed lack of influence of lactate on myoglobin redox stability at meat condition suggests that the color stability of lactate-enhanced fresh meat is not due to direct interactions between the ingredient and the heme protein.

Isolation and Characterization of Chelonia mydas Myoglobin

Green sea turtle – Chelonia mydas is a lung respiration animal that is able to dive and stay uder sea water for hours without needing to surface for oxygen. As oxygen supply in muscles is assured by myoglobin, we propose to study some characteristic of this muscle protein in green sea turtles. To achieve this objective, pure green sea turtle myoglobin has to be made available. Therefore, our first task is to purify this muscle protein from green sea turtles. Skeletal muscles from 3 green sea turtle hatchlings were studied microscopically and biochemically. Microscopy observation showed a general structure of striated muscle. Biochemical studies revealed that green sea turtle myoglobin could be more purely isolated to a certain degree by Sephadex G-75 gel filtration and purified by immunoaffinity gel chromatography rather than direct purification by DEAE-Sepharose ion exchange chromatography technique. SDS-PAGE analysis showed that green sea turtle myoglobin ran together with horse myoglobin as 17 kDa molecular weight proteins.

Assessment of the functional diversity of human myoglobin

Myoglobin is presumably the most studied protein in biology. Its functional properties as a dioxygen storage and facilitator of dioxygen transport are widely acknowledged. Experimental evidence also implicates an essential role for myoglobin in the heart in regulating nitric oxide homeostasis. Under normoxia, oxygenated myoglobin can scavenge excessive nitric oxide, while under hypoxia, deoxygenated myoglobin can reduce nitrite, an oxidative product of nitric oxide, to bioactive nitric oxide. Myoglobin-driven nitrite reduction can protect the heart from ischemia and reperfusion injury. While horse and mouse myoglobin have been previously described to reduce nitrite under these conditions, a comparable activity has not been detected in human myoglobin. We here show that human myoglobin is a fully functional nitrite reductase. To study the role of human myoglobin for nitric oxide homeostasis we used repeated photometric wavelength scans and chemiluminescence based experiments. The results revealed that oxygenated human myoglobin reacts with nitrite-derived nitric oxide to form ferric myoglobin and that deoxygenated human myoglobin acts as a nitrite reductase in vitro and in situ. Rates of both nitric oxide scavenging and nitrite reduction were significantly higher in human compared to horse myoglobin. These data extend the existing knowledge about the functional properties of human myoglobin and are the basis for further translational studies towards the importance of myoglobin for nitric oxide metabolism in humans.