Heme Group In Myoglobin Research Articles

A new non-enzymatic biosensor for the determination of bisphenol-A.

In this study, a new non-enzymatic carbon paste biosensor was developed for the determination of Bisphenol-A (BPA) based on Multiwalled Carbon Nanotube (MWCNT) modified Myoglobin (Mb). The measurement principle of the biosensor was developed based on the inhibition effect of BPA on the heme group of myoglobin in the presence of hydrogen peroxide. With the designed biosensor, measurements were taken in the potential range of (-0.15V & +0.65V) using the differential pulse voltammetry (DPV) method in the medium containing K4[Fe(CN)6]. The linear range for BPA was determined to be 100-1000µM. Response time was calculated as 16s. The limit of detection was set at 89μM. As a result, it has been proven that MWCNT modified myoglobin based biosensor is an alternative method that can be used for BPA determination, giving very sensitive and fast results.

Nonbonded Atomic Contacts Drive Ultrafast Helix Motions in Myoglobin.

The association and dissociation of small ligands regulate the functions of proteins through structural changes in the protein. Such structural changes propagate long distances, and this allostery plays a key role in molecular functions. However, the mechanism by which structural changes are transmitted is poorly understood. Here we show that nonbonded atomic contacts play an essential role in driving the displacement of a helix in picosecond time scale primary structural changes following the dissociation of carbon monoxide from the heme group in myoglobin. The present time-resolved ultraviolet resonance Raman study revealed that the amplitude of this helix displacement was reduced upon substitution of Val68, which contacts the heme in wild-type myoglobin, with a less bulky side chain (Ala). Our findings provided the first direct evidence that structural changes are transmitted not only by covalent bonds, salt bridges and hydrogen bonds but also by nonbonded atomic contacts in the primary protein response upon ligand dissociation. Furthermore, the present results indicate the importance of dense atomic packing in a protein structure for responding to the association and dissociation of small molecules. The high compactness of protein structures makes possible the propagation of structural changes, providing useful clues to the design of molecular machines.

Macromolecular crowding induces molten globule state in the native myoglobin at physiological pH

Here, we report the formation of molten globule state of the native myoglobin in crowded environment. We have used Soret absorption spectroscopy and far-UV circular dichroism to monitor changes in tertiary and secondary structures of myoglobin, respectively. Our results reveal that in the presence of ficoll 70, the secondary structure of myoglobin remains unchanged while tertiary structure is lost significantly. 1-anilinonaphthalene-8-sulfonate binding experiments showed that myoglobin in the presence of various concentrations of ficoll 70, has newly exposed hydrophobic surfaces. Dynamic light scattering measurements show that there is almost 1.5 times increase in the hydrodynamic volume of myoglobin in the crowded environment. These structural characteristics of myoglobin in the presence of 300mg/ml ficoll 70 resemble those of molten globule state. Isothermal titration calorimetric (ITC) measurements show that ficoll 70 binds to myoglobin, whereas it shows no interaction with apo form of the protein. ITC results indicate that the reason behind this unique behavior of ficoll 70 towards myoglobin may be interaction of ficoll 70 with the heme group of myoglobin, which was further confirmed by the docking studies. We hypothesize that the soft interactions between heme and ficoll 70 leads to the formation of molten globule in myoglobin.

Characterization of intermediate state of myoglobin in the presence of PEG 10 under physiological conditions

The macromolecular crowding progressively has been gaining prominence in recent years as it acts as a sword with double-edge on protein stability and folding, i.e., showing assorted results of having both stabilizing and destabilizing effects. We studied the effects of different concentrations of polyethylene glycol (PEG-10) on structure and stability of myoglobin. The tertiary structure of myoglobin was found to be perturbed in the presence of polyethylene glycol, however there was insignificant change in the secondary structure. It was observed that polyethylene glycol induces molten globule state in myoglobin, where the intermediate state holds hydrophobic patches and larger hydrodynamic volume as compared to the native protein. In addition, isothermal titration calorimetry (ITC) showed strong binding between myoglobin and polyethylene glycol, at the physiological pH. We hypothesize that polyethylene glycol induces molten globule conformation in myoglobin by interacting with heme group of myoglobin. We caution that the binding of protein with crowder and other soft interactions need to be gravely well thought-out when studying macromolecular crowding. In our case, destabilizing protein-crowder interactions could compete and overcome the stabilizing exclusion volume effect.

Gold nanoparticles-reduced graphene oxide based electrochemical immunosensor for the cardiac biomarker myoglobin

A composite consisting of gold nanoparticles and reduced graphene oxide (AuNPs@rGO) was electrochemically prepared in-situ on a screen printed electrode (SPE) which then was used as an immunosensor for the cardiac biomarker myoglobin. The nanocomposite was characterized by transmission electron microscopy (TEM, scanning electron microscopy (SEM), atomic force microscopy, FTIR and electrochemical impedance spectroscopy. For FTIR, TEM and SEM, the deposition was done on indium tin oxide coated glass plates. The immunosensor was obtained by immobilization of in-house generated antibody against cardiac myoglobin on the electrode surface. The immunosensing response was monitored using differential pulse voltammetry, which showed a reduction peak at ~ −0.5 V (vs. Ag/AgCl). The reduction peak arises from the reduction of iron moiety present in the heme group of myoglobin. The immunosensor exhibited dynamic linearity range from 1 ng.mL−1 to 1400 ng.mL−1 with the detection limit of ~0.67 ng.mL−1 for cardiac myoglobin. The obtained result was almost eight times better (in terms of detection limit) than that obtained with ELISA tests (with detection limit of ~4 ng.mL−1) using the same antibodies. The immunosensor was applied to analyze spiked serum samples also.

Electrochemical probing of the solution pH-induced structural alterations around the heme group in myoglobin

Protein folding-unfolding has received considerable attention because of its fundamental importance in the manifestation of biological functions, in molecular diagnostics, in drug development, and its close relationship to human health. Although many attempts have been made to study and monitor the protein unfolding, a facile and effective method for probing the structural changes of proteins is still highly desired. This work reports electrochemical voltammetry for probing the structural alterations around the heme group in myoglobin (Mb) induced by solution pH. This approach is based on the changes of the electron transfer (ET) signals of Mb upon the structural alteration because the heme group is deeply buried in the hydrophobic pocket in neutral conditions (in the native state), resulting in poor ET signals, while it dissociates from the hydrophobic pocket and becomes exposed to the solution in acidic and basic conditions (in the unfolding state) due to the breakage of the Fe-His93 bond, causing an obvious enhancement in ET signals. The good consistency of the results obtained with our electrochemical method with those of spectroscopic measurements (such as UV-vis, CD, and Raman spectroscopy) and molecular dynamics (MD) simulations substantially validates that our proposed method is a facile and effective way for probing structural changes around the heme group in Mb. Compared with the existing methods used for the study of structural changes of protein, our method demonstrated here could be advantageous in terms of its sensitivity, ease of operation, cost-effective feature, simple use of bare electrodes, and availability of abundant dynamic information. This study essentially offers a facile and effective electrochemical route to probe the structural changes around the heme group and the conformation alterations of heme-containing proteins.

Natural bond orbital analysis in the ONETEP code: Applications to large protein systems

First principles electronic structure calculations are typically performed in terms of molecular orbitals (or bands), providing a straightforward theoretical avenue for approximations of increasing sophistication, but do not usually provide any qualitative chemical information about the system. We can derive such information via post-processing using natural bond orbital (NBO) analysis, which produces a chemical picture of bonding in terms of localized Lewis-type bond and lone pair orbitals that we can use to understand molecular structure and interactions. We present NBO analysis of large-scale calculations with the ONETEP linear-scaling density functional theory package, which we have interfaced with the NBO 5 analysis program. In ONETEP calculations involving thousands of atoms, one is typically interested in particular regions of a nanosystem whilst accounting for long-range electronic effects from the entire system. We show that by transforming the Non-orthogonal Generalized Wannier Functions of ONETEP to natural atomic orbitals, NBO analysis can be performed within a localized region in such a way that ensures the results are identical to an analysis on the full system. We demonstrate the capabilities of this approach by performing illustrative studies of large proteins--namely, investigating changes in charge transfer between the heme group of myoglobin and its ligands with increasing system size and between a protein and its explicit solvent, estimating the contribution of electronic delocalization to the stabilization of hydrogen bonds in the binding pocket of a drug-receptor complex, and observing, in situ, the n → π* hyperconjugative interactions between carbonyl groups that stabilize protein backbones.

Ultrafast Dynamics of Resonance Energy Transfer in Myoglobin: Probing Local Conformation Fluctuations

We report here our systematic characterization of resonance energy transfer between intrinsic tryptophan and the prosthetic heme group in myoglobin in order to develop a novel energy-transfer pair as a molecular ruler in heme proteins to study local conformation fluctuations. With site-directed mutagenesis, we designed four tryptophan mutants along the A-helix of myoglobin and each mutant contains only a single tryptophan-heme energy-transfer pair. With femtosecond resolution, we observed, even at separation distances of 15-25 A, ultrafast energy transfer in tens to hundreds of picoseconds. On these time scales, the donor and acceptor cannot be randomized and the orientation factor in Forster energy transfer is highly restricted. Thus, direct measurement of the orientation-factor changes at different mutation sites reveals relative local structure flexibility and conformation fluctuations as particularly demonstrated here for positions of tryptophan 7 and 14. More importantly, the local environment relaxation occurs on the similar time scales of the energy transfer dynamics, resulting in a nonequilibrium dynamic process. With femtosecond- and wavelength-resolved fluorescence dynamics, we are able to determine the time scales of such nonequilibrium energy-transfer dynamics and elucidate the mechanism of the nonexponential energy-transfer dynamics caused by local dynamic heterogeneity and/or local environment relaxation.

The Nernst equation applied to oxidation–reduction reactions in myoglobin and hemoglobin. Evaluation of the parameters

Analyses of the binding of oxygen to monomers such as myoglobin employ the Mass Action equation. The Mass Action equation, as such, is not directly applicable for the analysis of the binding of oxygen to oligomers such as hemoglobin. When the binding of oxygen to hemoglobin is analyzed, models incorporating extensions of mass action are employed. Oxidation-reduction reactions of the heme group in myoglobin and hemoglobin involve the binding and dissociation of electrons. This reaction is described with the Nernst equation. The Nernst equation is applicable only to a monomeric species even if the number of electrons involved is greater than unity. To analyze the oxidation-reduction reaction in a molecule such as hemoglobin a model is required which incorporates extensions of the Nernst equation. This communication develops models employing the Nernst equation for oxidation-reduction reactions analogous to those employed for hemoglobin in the analysis of the oxygenation (binding of oxygen) reaction.

Classical Molecular Electrostatics: Recognition of Ligands in Proteins and the Vibrational Stark Effect

It is shown that classical electrostatics quantitatively describes both the binding of the diatomic ligands XO (X = C, N, O) to the heme group in myoglobin and the dependence of their vibrational frequencies upon an external field, the vibrational Stark effect. The key is a proper treatment of induced dipoles. The results suggest that ligand binding occurs via an "electrostatic bond", a generalization of the standard ionic bond to include induction, and, more generally, that classical electrostatics can replace quantum mechanics for a considerable simplification of some complex problems.

Embedding a Reporter

CHEMISTRY The nitrile (CN) group can be a useful infrared reporter in proteins because it has a strong stretching vibration near ∼2200 cm−1, a spectral region usually free from interfering absorptions in a biochemical environment. Schultz et al. have devised a protocol to introduce the non-naturally occurring amino acid para- cyano-l-phenylalanine (pCNPhe) into proteins during bacterial synthesis, using an orthogonal nonsense suppressor transfer RNA (tRNA) paired with an aminoacyl tRNA synthetase derived from Methanococcus janannaschi. They apply this system to incorporate the pCNPhe reporter in place of a histidine residue (His64) near the ligand-binding site of the heme group in myoglobin. When water was bound in the active site, they observed an 11-cm−1 shift in frequency relative to pCNPhe absorption in pure water or buffer solution, a change consistent with increased water polarity in the binding pocket. Changes in the observed CN stretching frequency were also consistent with the bent conformations of Fe(II)-bound NO and O2, as well as the linear CO-Fe(II) complex. — PDS J. Am. Chem. Soc. 128 , 10.1021/ja0636690 (2006).

Functional Probing of Arginine Residues in Proteins Using Mass Spectrometry and an Arginine-Specific Covalent Tagging Concept

The reactivity of arginine residues in model proteins (ubiquitin, cytochrome c, myoglobin, ribonuclease A, lysozyme) was examined using a selective tagging reaction in combination with on-line monitoring of the reaction progress by electrospray ionization mass spectrometry (ESI-MS). The kinetics of this reaction, based on the cyclization of the guanidine group of arginine with 2,3-butanedione and phenylboronic acid at pH 8-10, allow the grouping of arginines in "exposed" or "partially buried" residues, because they differ substantially in their reaction rate constants for the conversion of the guanidine groups. The method allows one to differentiate between different protein conformations as shown for myoglobin and its apo form and native and reduced ribonuclease A: Removal of the heme group in myoglobin resulted in an increased reactivity for the two partially buried arginines. For RNAse A, quantitative reduction of the disulfide bonds lead to the exposure of an additional arginine residue and two different conformations of the reduced protein were observed by ESI-MS that could be distinguished according to their charge-state distribution. Experimentally obtained accessibilities were compared with solvent-accessibility data calculated from 3D structures and substantial agreement between both techniques was observed.

Desferrioxamine Inhibits Production of Cytotoxic Heme to Protein Cross-Linked Myoglobin: A Mechanism to Protect against Oxidative Stress without Iron Chelation

The heme group of myoglobin can form a covalent bond to the protein when met (ferric) myoglobin is reacted with peroxides under acidic conditions. This heme to protein cross-linked species is highly pro-oxidant and found in the urine of patients with rhabdomyolytic-associated acute renal failure. Desferrioxamine, an iron-chelating agent used in the treatment of iron overload, is reported to be partially effective at preventing kidney failure following rhabdomyolysis. In this article, we show that in addition to its capacity as an iron chelator, desferroxamine can inhibit the peroxide-induced formation of heme to protein cross-linked myoglobin and decreases the pro-oxidant activity of both native and heme to protein cross-linked myoglobin. The mechanism of peroxidation and of heme to protein cross-linking involves the formation of ferryl intermediate (Fe(4+)=O(2-)), and it is by the reduction of this intermediate to the ferric form that desferrioxamine can exert inhibitory effects. The concentrations at which desferrioxamine inhibits the formation of heme to protein cross-linked myoglobin and prevents the pro-oxidant activity of native and oxidatively modified myoglobins are comparable to the concentrations used for in vivo studies of iron-related oxidative stress. Thus, the ameliorative effects of treatment of posthemolytic events by desferrioxamine cannot be exclusively assigned to its ability to chelate free iron.

Immunogenicity of Heme Complexes of Peptides Designed to Mimic the Heme Environment of Myoglobin and Hemoglobin

In the preceding paper (Protein J. 25, pages 37-49, 2005), we reported the preparation and oxygen-binding properties of peptides that form stable complexes with heme mimic. The design of the peptides was based on the natural environment of the heme group in myoglobin (Mb) and in the alpha- and beta-subunits of human adult hemoglobin (Hb). In the present work, the heme-peptides were each administered into mice, either as emulsions in adjuvant (both for injections and boosters) or intravenously as solutions in phosphate-buffered saline. Antibody (Ab) responses, monitored up to 14 weeks after the first administration, showed that when the heme-peptides were injected with adjuvant they stimulated Ab responses against the immunizing peptide, which in most cases bound to the correlate protein (Mb or Hb). However these heme-peptides were non-immunogenic when administered in PBS intravenously. It is concluded that heme-peptides:(a) would not trigger an adverse immune response if used for transfusion purposes.

Oxygen-binding Heme Complexes of Peptides Designed to Mimic the Heme Environment of Myoglobin and Hemoglobin

Development of effective resuscitation agents for blood-loss replacement in trauma or surgery is extremely important despite substantial improvements in screening methods of blood from human donors. This paper reports the design and synthesis of peptides that mimic the natural environment of the heme group in myoglobin (Mb) and in the alpha- and beta-subunits of human adult hemoglobin (Hb). The designs were based on the fact that the heme group in the aforementioned proteins is sandwiched between helices E and F. Fifteen test peptides and six control peptides were synthesized, and their ability to form stable complexes with heme was investigated. It was found that none of the control peptides or proteins was able to bind heme. However, each of the peptides that were designed to mimic the E-F helices, and even shorter designs, which removed from this region residues that do not contribute to contacts with the heme group, were each able to bind one mole of heme per mole of peptide forming peptide heme complexes that were stable to manipulation and behaved as single molecular species. Oxygen binding measurements on the reduced peptide-heme complexes showed that these compounds bind oxygen and give visible spectra that were typical of oxygenated heme-proteins. In oxygen binding measurements done under different partial pressures of oxygen, the heme peptide complexes gave hyperbolic oxygen-saturation curves, but showed slight differences in their P50 values. The P50 values ranged from 3.8 mmHg for the heme peptide B7 complex to 13.7 mmHg for the heme peptide D13 complex (under the same conditions, P50 values for Hb and Mb were 34.0 and 5.5 mmHg, respectively). It is concluded that peptide constructs designed to mimic the heme-binding regions of Mb or the Hb subunits were able to form coordinate 1:1 complexes with heme, and these complexes bind oxygen in a manner expected for single subunit heme proteins.

Fiber-optic immunosensor for measurement of myoglobin

A self-contained fiber-optic immunosensor was developed to measure the 16,500-Da protein myoglobin. The sensing element was constructed by entrapment of Cascade Blue-labeled antibody within polyacrylamide gel at the distal face of an optical fiber 300 microns in core diameter. The polyacrylamide gel composition was optimized to allow diffusion of myoglobin but to exclude hemoglobin and higher-molecular-mass proteins from the sensing area. The analytical signal was derived from fluorescence energy transfer between Cascade Blue and the heme group of myoglobin. Fluorescence quenching occurred when myoglobin bound to labeled antibody. The total amount of fluorescence quench was dependent on the antibody labeling conditions and the amount of antibody incorporated in the sensor gel matrix. Myoglobin concentrations > 5 nmol/L (83 micrograms/L) were measurable with response times of 15 to 130 min limited by diffusion into the sensing element. This report demonstrates the technical feasibility for a self-contained immunosensor to measure a protein analyte.

Molecular and stochastic dynamics of proteins.

The rapid fluctuations of protein atoms derived from molecular dynamics simulations can be extrapolated to longer-time motions by effective single-particle stochastic models. This is demonstrated by an analysis of velocity autocorrelation functions for the atoms of lysine side chains in the active site of RNase A. The atomic motions are described by a bounded stochastic model with the friction and noise parameters determined from a molecular dynamics simulation. The low-frequency relaxation behavior is shown to result from collisional damping rather than dephasing. Extrapolation of these results to the quasistochastic motion of the heme group in myoglobin provides an explanation of 57Fe Mössbauer spectroscopic data.

Generalized moment expansion for observables of stochastic processes in dimensions d>1: Application to Mössbauer spectra of proteins

The generalized moment expansion provides an effective algorithm for the approximation of the time dependence of observables that monitor stochastic processes. Up to now this method had been applied mainly to one-variable birth–death processes or to one-dimensional Fokker–Planck systems since in these cases analytical and numerical methods for the evaluation of the generalized moments were available. Here we demonstrate that numerical sparse matrix methods can be used to extend the range of application of the generalized moment expansion to higher dimensions. For this purpose we introduce a simple but general discretization scheme for Fokker–Planck operators of Smoluchowski type which is, for these special operators, superior to common numerical discretization schemes for differential operators. As an application we determine the Mössbauer absorption spectrum of a Brownian particle in certain two- and three-dimensional potentials. This serves as a model for the motion of the heme group in myoglobin.

Magnesium porphyrin-globin and -amino acid complexes

Complexes of magnesium mesoporphyrin (MgMP) and magnesium protoporphyrin (MgPP) with apomyoglobin and apohemoglobin, and amino acid species of the type MgMP/PP (amino acid) 2 have been studied by electronic, circular dichroism (CD) and optical rotatory dispersion (ORD) spectroscopy [1−3]. In the case of the protein complexes, specific spectral differences were observed for the myoglobin (Mb) and hemoglobin (Hb) products. These have been interpreted in terms of the formation of six-coordinate magnesium aquo species, Mg(porphyrin)(-histidine)(H 2O), for MgMPMb and MgPPMb and five-coordinate, Mg(porphyrin)(-histidine), species for MgMPHb and MgPPHb [1]. These results highlight the importance of relatively small differences in the protein environment of the heme group in myoglobin and hemoglobin, on binding at the sixth coordination site. It is possible that a favourable hydrogen bonding interaction with the distal imidazole group (of the type recently described for oxy Mb [4] and CO heme proteins [5]) may stabilize the binding of a water molecule to Mg in the Mb species. ▪ Aqueous solutions of the magnesium porphyrins containing the chiral amino acids, L-histidine, L-serine, L-threonine and L-proline, produce prominent induced Cotton effects [1, 2]. However the CD/ORD spectra of these species differ from those of the magnesium porphyrin Mb and Hb protein complexes. Electronic band positions indicate the complexes producing the Cotton effects to be six-coordinate Mg(porphyrin)(amino acid) 2 entities, containing two amino acids bound to the metal rather than only one amino acid residue as for the protein species. The electronic spectra also indicate the presence of five-coordinate (Mg(porphyrin)(amino acid) species in the solutions but these do not produce Cotton effects. Induced Cotton effects of the magnitude observed for the Mg(porphyrin)(amino acid) 2complexes presumably require reasonable firm stereochemical location of the coordinated amino acids. For L-histidine we suggest the ligands are localized primarily by ππ bonding interactions between the ligand aromatic π system and the d π orbitals of Mg [2]. For the similar amino acids, L-serine and L-threonine, molecular models indicate that hydrogen bonding between the ligand −OH groups and the porphyrin COO − side chains fix these ligands in well-defined positions. L-proline is a special case in that binding through the ring nitrogen atom produces a new chiral centre at that atom. Interestingly, steric interactions between proline and the porphyrin ring determine the chirality of nitrogen to be opposite to that at the asymmetric carbon atom (as illustrated in Fig. 1). Inversion of this type occurs for a similar reason in the case of proline complexes Cu 2+ [6].