Showing papers on "Myoglobin published in 2000"
TL;DR: It is shown that below 180 K photodissociated ligands migrate to specific sites within an internal cavity—the distal haem pocket— of an essentially immobilized, frozen protein, from where they subsequently rebind by thermally activated barrier crossing.
Abstract: Myoglobin, a small globular haem protein that binds gaseous ligands such as O2, CO and NO reversibly at the haem iron, serves as a model for studying structural and dynamic aspects of protein reactions. Time-resolved spectroscopic measurements after photodissociation of the ligand revealed a complex ligand-binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein1,2,3. To observe the structural changes induced by ligand dissociation, we have carried out X-ray crystallographic investigations of carbon monoxy-myoglobin (MbCO mutant L29W) crystals illuminated below and above 180 K, complemented by time-resolved infrared spectroscopy of CO rebinding. Here we show that below 180 K photodissociated ligands migrate to specific sites within an internal cavity—the distal haem pocket—of an essentially immobilized, frozen protein, from where they subsequently rebind by thermally activated barrier crossing. Upon photodissociation above 180 K, ligands escape from the distal pocket, aided by protein fluctuations that transiently open exit channels. We recover most of the ligands in a cavity on the opposite side of the haem group.
382 citations
TL;DR: The 1.4 Å resolution crystal structure of a ligand-binding intermediate in carbonmonoxy myoglobin is reported that may have far-reaching implications for understanding the dynamics of ligand binding and catalysis.
Abstract: Small molecules such as NO, O2, CO or H2 are important biological ligands that bind to metalloproteins to function crucially in processes such as signal transduction, respiration and catalysis. A key issue for understanding the regulation of reaction mechanisms in these systems is whether ligands gain access to the binding sites through specific channels and docking sites, or by random diffusion through the protein matrix. A model system for studying this issue is myoglobin, a simple haem protein. Myoglobin has been studied extensively by spectroscopy, crystallography, computation and theory1,2,3,4,5,6,7,8,9,10,11. It serves as an aid to oxygen diffusion but also binds carbon monoxide, a byproduct of endogenous haem catabolism. Molecular dynamics simulations3,4,5, random mutagenesis6 and flash photolysis studies7,8,9,10 indicate that ligand migration occurs through a limited number of pathways involving docking sites. Here we report the 1.4 A resolution crystal structure of a ligand-binding intermediate in carbonmonoxy myoglobin that may have far-reaching implications for understanding the dynamics of ligand binding and catalysis.
231 citations
TL;DR: Myoglobin content and body mass were both positively and significantly correlated to maximum dive duration in odontocetes; this differed from the relationship for mysticetes.
Abstract: Cetaceans exhibit an exceptionally wide range of body mass that influence both the capacities for oxygen storage and utilization; the balance of these factors is important for defining dive limits. Furthermore, myoglobin content is a key oxygen store in the muscle as it is many times higher in marine mammals than terrestrial mammals. Yet little consideration has been given to the effects of myoglobin content or body mass on cetacean dive capacity. To determine the importance of myoglobin content and body mass on cetacean diving performance, we measured myoglobin content of the longissimus dorsi for ten odontocete (toothed whales) and one mysticete (baleen whales) species ranging in body mass from 70 to 80 000 kg. The results showed that myoglobin content in cetaceans ranged from 1.81 to 5.78 g (100 g wet muscle) −1 . Myoglobin content and body mass were both positively and significantly correlated to maximum dive duration in odontocetes; this differed from the relationship for mysticetes. Overall, the combined effects of body mass and myoglobin content accounts for 50% of the variation in cetacean diving performance. While independent analysis of the odontocetes showed that body mass and myoglobin content accounts for 83% of the variation in odontocete dive capacity.
188 citations
TL;DR: H-FABP is more sensitive than both myoglobin and CK-MB, more specific than myoglobin for detecting AMI within 12 hours after the onset of symptoms, and shows the highest values for both diagnostic efficacy and ROC curve analysis.
Abstract: Heart-type fatty acid-binding protein (H-FABP) is a low molecular weight cytoplasmic protein and present abundantly in the myocardium. When the myocardium is injured, as in the case of myocardial infarction, low molecular weight cytoplasmic proteins including H-FABP are released into the circulation and H-FABP is detectable in a blood sample. We have already developed a direct sandwich-ELISA for quantification of human H-FABP using two distinct types of monoclonal antibodies specific for human H-FABP. In this study we investigated the clinical validity of H-FABP as a biochemical diagnostic marker in the early phase of acute myocardial infarction (AMI). To evaluate the diagnostic usefulness of H-FABP in the early phase of AMI, blood samples were obtained from the following patients within 12 hours after the appearance of symptoms, and serum levels of H-FABP were compared with those of conventional diagnostic markers, such as myoglobin and creatine kinase isoenzyme MB (CK-MB). Blood samples were collected from patients with confirmed AMI (n=140), patients with chest pain who were afterwards not classified as AMI by normal CK-MB levels (non-AMI) (n=49) and normal healthy volunteers (n=75). The serum concentration of H-FABP was quantified with our direct sandwich-ELISA. The concentration of myoglobin mass was measured with a commercial RIA kit. The serum CK-MB activity was determined with an immuno-inhibition assay kit. The overall sensitivity of H-FABP, within 12 hours after the appearance of symptoms, was 92.9%, while it was 88.6% with myoglobin and 18.6% with CK-MB. The overall specificity of H-FABP was 67.3%, while it was 57.1% with myoglobin and 98.0% with CK-MB. The diagnostic efficacy rates with these markers were 86.2% (H-FABP), 80.4% (myoglobin) and 39.2% (CK-MB), respectively. The diagnostic validity of H-FABP was further assessed by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) of H-FABP was 0.921, which was significantly greater than with myoglobin (AUC: 0.843) and CK-MB (AUC: 0.654). These parameters, such as sensitivity, specificity, diagnostic efficacy and diagnostic accuracy, obtained for patients with chest pain within 3 hours and/or 6 hours after the onset of symptoms were almost the same as those for patients within 12 hours after symptoms. H-FABP is more sensitive than both myoglobin and CK-MB, more specific than myoglobin for detecting AMI within 12 hours after the onset of symptoms, and shows the highest values for both diagnostic efficacy and ROC curve analysis. Thus, H-FABP has great potential as an excellent biochemical cardiac marker for the diagnosis of AMI in the early phase.
178 citations
TL;DR: Sarcoplasmic protein diffusion is strongly impaired by the presence of the myofilamental lattice, which also gives rise to differences in diffusivity between different fiber types and this molecular sieve property progressively reduces intracellular diffusion of proteins when the molecular diameter increases to more than approximately 10 nm.
162 citations
TL;DR: The design and engineering of a copper-binding site in sperm whale myoglobin (swMb) is reported here based on structural comparison and computer modeling of swMb and CcO.
Abstract: Myoglobins (Mb) are small globular heme proteins that serve as O2 carriers. Heme-copper oxidases (HCOs) are large membrane-bound proteins that catalyze proton-coupled reduction of O2 to water. Mb contains a single heme center, while HCOs contain a high-spin heme-CuB dinuclear center, a low-spin heme center, and in certain subclasses of HCO enzymes such as cytochrome c oxidases (CcO) a dinuclear copper center called CuA. While Mb is one of the most well-characterized proteins, many questions about the structure and function of HCOs, such as the role of the CuB center, the origin of spin coupling between CuB and heme, and the exact nature of the reaction intermediates, remain to be fully understood. We report here the design and engineering of a copper-binding site in sperm whale myoglobin (swMb) based on structural comparison and computer modeling of swMb and CcO. UV−vis studies of the resting state of the designed protein swMb((L29H, F43H) (called CuBMb) suggest that a single copper-binding site is create...
155 citations
TL;DR: The experiment shows that the pathway of a small molecule in its trajectory through a protein may be modified by site-directed mutagenesis, and that migration within the protein matrix to the active site involves a limited number of pre-existing cavities identified in the interior space of the protein.
Abstract: We determined the structure of the photolytic intermediate of a sperm whale myoglobin (Mb) mutant called Mb-YQR [Leu-(B10)→Tyr; His(E7)→Gln; Thr(E10)→Arg] to 1.4-A resolution by ultra-low temperature (20 K) x-ray diffraction. Starting with the CO complex, illumination leads to photolysis of the Fe–CO bond, and migration of the photolyzed carbon monoxide (CO*) to a niche in the protein 8.1 A from the heme iron; this cavity corresponds to that hosting an atom of Xe when the crystal is equilibrated with xenon gas at 7 atmospheres [Tilton, R. F., Jr., Kuntz, I. D. & Petsko, G. A. (1984) Biochemistry 23, 2849–2857]. The site occupied by CO* corresponds to that predicted by molecular dynamics simulations previously carried out to account for the NO geminate rebinding of Mb-YQR observed in laser photolysis experiments at room temperature. This secondary docking site differs from the primary docking site identified by previous crystallographic studies on the photolyzed intermediate of wild-type sperm whale Mb performed at cryogenic temperatures [Teng et al. (1994) Nat. Struct. Biol. 1, 701–705] and room temperature [Srajer et al. (1996) Science 274, 1726–1729]. Our experiment shows that the pathway of a small molecule in its trajectory through a protein may be modified by site-directed mutagenesis, and that migration within the protein matrix to the active site involves a limited number of pre-existing cavities identified in the interior space of the protein.
146 citations
TL;DR: The femtosecond coherence spectroscopy (FCS) was applied to the heme protein myoglobin in this article, where the relative phase and amplitude of the Fe−His vibration associated with the sole covalent linkage between heme and the protein were analyzed as a function of sample state and pump/probe carrier frequency.
Abstract: The technique of femtosecond coherence spectroscopy (FCS) is applied to the heme protein myoglobin. Photostable samples of deoxy myoglobin (Mb) and photochemically active samples of the nitric oxide adduct (MbNO) are investigated. The pump-induced change in the probe transmittance for both samples displays coherent oscillations that, when transformed into the frequency domain, are in agreement with the resonance Raman spectrum of deoxy Mb. This indicates that the coherences associated with the photoreactive sample (MbNO) arise from the rapidly changing forces appearing in the crossing region(s) between the reactant and product state potential energy surfaces. The relative phase and amplitude of the Fe−His vibration, associated with the sole covalent linkage between the heme and the protein, are analyzed as a function of sample state and pump/probe carrier frequency. The dependence of the phase on carrier frequency is found to be significantly different for the “field driven” coherence in Mb and the “react...
100 citations
TL;DR: The viewpoint that pre-existing 'packing defects' in the protein interior play a major role in controlling the dynamics of ligand binding, including oxygen, and thereby acquire a survival value is supported.
98 citations
96 citations
TL;DR: The magnitude and salt dependence of interactions between ionizable groups were exaggerated in pK(a) calculations with the finite-difference Poisson-Boltzmann method applied to a static structure, even when the protein interior was treated with arbitrarily high dielectric constants.
TL;DR: It is demonstrated that IR spectroscopic studies of protein aggregation using a combination of thermal and chemical denaturing factors could provide a means to populate and characterize aggregation intermediates.
TL;DR: The data indicate that reaction of wild-type human Mb and H2O2 differs from the corresponding reaction of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolecular disulfide bond formation.
TL;DR: Kinetic studies of the peroxynitrite-mediated oxidations of oxymyoglobin and oxyhemoglobin showed that the mechanisms of these reactions are more complex than what had previously been reported; both reactions proceed in two steps.
Abstract: Kinetic studies of the peroxynitrite-mediated oxidations of oxymyoglobin (MbFeO(2)) and oxyhemoglobin (HbFeO(2)) showed that the mechanisms of these reactions are more complex than what had previously been reported; both reactions proceed in two steps. For myoglobin, we found that the small amount of deoxymyoglobin (MbFe(II)) which is in equilibrium with MbFeO(2) is first oxidized by peroxynitrous acid to ferryl myoglobin (MbFe(IV)=O). Then, in the second step, MbFe(IV)=O is reduced by peroxynitrous acid to metmyoglobin (metMb). The second-order rate constant values obtained at pH 7.3 and 20 degrees C for the two steps are (5.4 +/- 0.2) x 10(4) and (2.2 +/- 0.1) x 10(4) M(-)(1) s(-)(1), respectively. Analogous studies with hemoglobin suggest that its reaction with peroxynitrite follows the same mechanism. In this case, the second-order rate constant values measured at pH 7.0 and 20 degrees C for the two steps are (8.8 +/- 0.4) x 10(4) and (9.4 +/- 0.7) x 10(4) M(-)(1) s(-)(1), respectively. A possible mechanism in the absence as well as in the presence of CO(2) and the relevance of these reactions in vivo are discussed.
TL;DR: Crystal structures of the reactive short-lived species that occur in chemical or binding reactions can be determined using X-ray crystallography via time-resolved or kinetic trapping approaches.
TL;DR: The location of the distal histidine in the active site provides a critical balance between the formation and subsequent decay of the oxo-ferryl porphyrin radical cation (compound I), a catalytic species for one- and two-electron oxidation and oxygen transfer reactions.
TL;DR: In this article, a model of carbon monoxide (CO) binding to myoglobin was presented based on extensive ab initio calculations of CO interacting with a model heme-imidazole group which was performed using a fully quantum mechanical Hartree-Fock/density functional theory (HF/DFT) hybrid method.
Abstract: We present a microscopic model of carbon monoxide (CO) binding to myoglobin which reproduces the experimentally observed Arrhenius pre-exponential factor of 109 s−1 and activation enthalpy distribution centered at 12 kJ/mol. The model is based on extensive ab initio calculations of CO interacting with a model heme-imidazole group which we performed using a fully quantum mechanical Hartree–Fock/density functional theory (HF/DFT) hybrid method. We fit the HF/DFT calculated energies, obtained for over 1000 heme-CO structures with varied CO and iron positions and orientations for both high (S=2) and low (S=0) spin states, to a model potential function which includes a bonding interaction in both of the spin states, electrostatic, and anisotropic Lennard-Jones-type interactions. By combining the x-ray determined protein structure with this potential and protein-CO interactions and internal heme interaction potentials obtained from established molecular dynamics literature, we calculate the energy required for the CO to reach the spin crossing from the heme pocket. We find that the transition between the two spin states occurs when CO and iron have activation enthalpies of 8 kJ/mol and 3 kJ/mol, respectively, which are necessary to move CO towards the iron and the iron atom relative to the heme plane Npyr. At the same time we find that 1 kJ/mol is needed to move Ne of His-64 and Cγ of Val-68 relative to the heme group. The requirement that these motions be synchronized reduces the Arrhenius pre-exponential by a factor of 150 from the 1012 s−1 obtained from CO motion across the heme pocket, leaving a factor of ∼ 6 to account for CO orientation and nonadiabaticity of the electronic spin change. The observed width of the enthalpy distribution is reproduced by assuming a Gaussian distribution of the heme positions with a standard deviation of 0.2 A. We characterize the conformational relaxation by calculating an enthalpy barrier using x-ray structures of myoglobin in both the MbCO photoproduct and deoxy conformations, and we find a small difference, ∼ 5 kJ/mol, between the two conformations.
TL;DR: First direct evidence of a hydrogen bond in both alpha- and beta-chains of oxyhemoglobin is reported, as revealed by heteronuclear NMR spectra of chain-selectively labeled samples, suggesting that NMR could be used to obtain very high-resolution heme pocket structures of Hb, Mb, and other heme proteins.
Abstract: Compared with free heme, the proteins hemoglobin (Hb) and myoglobin (Mb) exhibit greatly enhanced affinity for oxygen relative to carbon monoxide. This physiologically vital property has been attributed to either steric hindrance of CO or stabilization of O2 binding by a hydrogen bond with the distal histidine. We report here the first direct evidence of such a hydrogen bond in both α- and β-chains of oxyhemoglobin, as revealed by heteronuclear NMR spectra of chain-selectively labeled samples. Using these spectra, we have assigned the imidazole ring 1H and 15N chemical shifts of the proximal and distal histidines in both carbonmonoxy- and oxy-Hb. Because of their proximity to the heme, these chemical shifts are extremely sensitive to the heme pocket conformation. Comparison of the measured chemical shifts with values predicted from x-ray structures suggests differences between the solution and crystal structures of oxy-Hb. The chemical shift discrepancies could be accounted for by very small displacements of the proximal and distal histidines. This suggests that NMR could be used to obtain very high-resolution heme pocket structures of Hb, Mb, and other heme proteins.
TL;DR: Ligand binding kinetics and resonance Raman spectroscopy are used to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands and suggest hydrogen bonding stabilization of bound oxygen.
Abstract: The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes.
TL;DR: In the uncharged agarose gel, diffusions of myoglobin are not effected by the change in pH and the ionic strength, indicating no electrostatic interaction between the gel and myoglobin, and in the negatively charged λ-carrageenan gel, the diffusion is accelerated by electrostatic attraction, but it is extensively hindered by the electrostatic repulsion.
Abstract: To study the effect of charge on protein diffusion in hydrogels, mutual diffusion of a globular protein, myoglobin, has been investigated at various pH and ionic strength levels in two kinds of polysaccharide gels, neutral agarose gel and anionic carrageenan gel, by the recently developed electronic speckle pattern interferometry method. In the uncharged agarose gel, diffusions of myoglobin are not effected by the change in pH and the ionic strength, indicating no electrostatic interaction between the gel and myoglobin. The experimental data in agarose gel agree with the combined model proposed by Clague and Philips for the diffusion of spheres in hydrogels. While in the negatively charged λ-carrageenan gel, the diffusion of myoglobin is accelerated by electrostatic attraction when the pH is lower than the isoelectric point (pI) of the protein, but it is extensively hindered by the electrostatic repulsion when pH > pI. The diffusion of myoglobin in λ-carrageenan gel agrees with the Tsai and Strieder model...
TL;DR: Investigation of the mechanism of interaction between GSNO and hemoglobin shows that GSNO reacts with deoxyHb to form glutathione, nitric oxide, and ferric hemoglobin, which provides a mechanism for how deoxygenation of hemoglobin/myoglobin could facilitate nitrosylated hemoglobin release from S-nitrosothiols and represents a potential physiological mechanism of S- nitrogenothiol metabolism.
TL;DR: Evidence is reported for nonspecific interactions between xenon and the exterior of myoglobin in aqueous solution, in addition to a previously reported internal binding interaction, that may permit the transfer of polarization between these nuclei via the nuclear Overhauser effect.
Abstract: The high sensitivity of the magnetic resonance properties of xenon to its local chemical environment and the large 129Xe NMR signals attainable through optical pumping have motivated the use of xenon as a probe of macromolecular structure and dynamics. In the present work, we report evidence for nonspecific interactions between xenon and the exterior of myoglobin in aqueous solution, in addition to a previously reported internal binding interaction. 129Xe chemical shift measurements in denatured myoglobin solutions and under native conditions with varying xenon concentrations confirm the presence of nonspecific interactions. Titration data are modeled quantitatively with treatment of the nonspecific interactions as weak binding sites. Using laser-polarized xenon to measure 129Xe spin-lattice relaxation times (T1), we observed a shorter T1 in the presence of 1 mM denatured apomyoglobin in 6 M deuterated urea (T1 = 59 ± 1 s) compared with that in 6 M deuterated urea alone (T1 = 291 ± 2 s), suggesting that nonspecific xenon-protein interactions can enhance 129Xe relaxation. An even shorter T1 was measured in 1 mM apomyoglobin in D2O (T1 = 15 ± 0.3 s), compared with that in D2O alone (T1 = 506 ± 5 s). This difference in relaxation efficiency likely results from couplings between laser-polarized xenon and protons in the binding cavity of apomyoglobin that may permit the transfer of polarization between these nuclei via the nuclear Overhauser effect.
TL;DR: The results show that the free amino group and tryptophan contents of the myoglobin-phenol derivatives decrease with the increasing extent to which the protein becomes derivatized, and the solubility of theMyoglobin- Phenol derivatives decreases in the pH range 3.5-6.5 as compared to solubilty of the native protein.
Abstract: This paper deals with the interactions of chlorogenic, caffeic, and quinic acids and p-quinone with myoglobin. The myoglobin derivatives formed have been characterized in terms of physicochemical properties and susceptibility to proteolysis. The results show that the free amino group and tryptophan contents of the myoglobin-phenol derivatives decrease with the increasing extent to which the protein becomes derivatized. Furthermore, the solubility of myoglobin-phenol derivatives decreases in the pH range 3.5-6.5 as compared to solubility of the native protein. The reaction also influences the hydrophilic-hydrophobic character of the protein. The isoelectric point of the derivatized myoglobin is shifted to a lower pH value, and formation of high molecular fractions is also documented. This paper also demonstrates the influence of the protein derivatization with plant phenols on susceptibility to digestion by trypsin, alpha-chymotrypsin, and pepsin, determined in vitro. The enzymatic digestion of the derivatized proteins is adversely affected.
TL;DR: Besides acting as an oxygen store during times of reduced blood oxygen supply, myoglobin can also facilitate intracellular oxygen transport by diffusion of oxymyoglobin along a PO(2) gradient by applying new findings on the intrace cellular diffusivity of myoglobin in a model calculation.
Abstract: Besides acting as an oxygen store during times of reduced blood oxygen supply, myoglobin can also facilitate intracellular oxygen transport by diffusion of oxymyoglobin along a Po2 gradient. We reassess the importance of myoglobin-facilitated oxygen diffusion by applying new findings on the intracellular diffusivity of myoglobin in a model calculation.
TL;DR: Physiological concentrations of ascorbic acid continuously provided by red blood cells probably rereduces Fe(III)P, leading to the apparent long lifetime of the dioxygenated species of FeP.
TL;DR: The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins in the presence of hydrogen peroxide.
Abstract: The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and protohemin-6(7)-L-histidine methyl ester (HM-H) were prepared by condensation of suitably protected Arg-Ala or His residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from horse heart, yielding the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectral, binding and catalytic properties of Mb-RA and Mb-H are significantly different from those of Mb. As shown by MM and MD calculations, these differences are determined by some local structural changes around the heme which are generated by increased mobility of a key peptide segment (Phe43-Lys47), containing the residue (Lys45) that in native Mb interacts with one of the porphyrin carboxylate groups. In the reconstituted Mbs this carboxylate group is bound to the Arg-Ala or His residue and is no longer available for electrostatic interaction with Lys45. The mobility of the peptide segment near the active site allows the distal histidine to come to a closer contact with the heme, and in fact Mb-RA and Mb-H exist as an equilibrium between a high-spin form and a major low-spin, six-coordinated form containing a bis-imidazole ligated heme. The two forms are clearly distinguishable in the NMR spectra, that also show that each of them consists of a mixture of the two most stable isomers resulting from cofactor reconstitution, as also anticipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide can displace the bound distal histidine, but their affinity is reduced. On the other hand, mobilization of the peptide chain around the heme in the reconstituted Mbs increases the accessibility of large donor molecules at the heme periphery, with respect to native Mb, where a rigid backbone limits access to the distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins in the presence of hydrogen peroxide.
TL;DR: The current evidence supports the role of Mb in the physical diffusion of O2; however, the unimpaired aerobic function of Mb knockout mice indicates that this role may not be critical to O2 supply in active muscle.
Abstract: Myoglobin (Mb) is a large protein that reversibly binds oxygen in the muscle cell and is thought to be critical for O2 supply to the mitochondria during exercise. The role of Mb in aerobic function is evaluated based on the physical properties of Mb as an O2 carrier and experimental evidence of Mb function in vivo. This role depends on the reversible binding of O2 by Mb depending on PO2, which results in: (1) storage of O2; (2) buffering of PO2 in the cell to prevent mitochondrial anoxia; and (3) parallel diffusion of O2 (so-called, 'facilitated diffusion'). The storage role is well established in diving mammals and buffering of cell PO2 above anoxic levels is shown here by in vivo magnetic resonance spectroscopy (MRS). However, the quantitative role of Mb in 'facilitated' or parallel diffusion of O2 is controversial. Evidence in support of this role is from MRS analyses, which reveal rapid Mb desaturation with exercise, and from the proportionality of Mb content of a muscle to the O2 diffusion limitation. Recent experiments with myoglobin knockout mice demonstrating high levels of aerobic function in normal and myoglobin-free mice argue against a link between Mb and oxidative phosphorylation. Thus, the current evidence supports the role of Mb in the physical diffusion of O2; however, the unimpaired aerobic function of Mb knockout mice indicates that this role may not be critical to O2 supply in active muscle.
TL;DR: The aim of this multicenter study was to compare H-FABP with myoglobin, cTnI, and CK-MB in the early detection of AMI in patients presenting with myocardial injury.
Abstract: Biochemical evidence of acute myocardial infarction (AMI) is delayed by the delay of appearance of serum cardiac markers in the blood after myocardial injury. Heart-type fatty acid-binding protein (H-FABP), a small (15 kDa) cytoplasmic protein (1) involved in lipid homeostasis, is abundant in heart muscle (2). H-FABP is ∼10-fold lower in skeletal muscle than in heart muscle, and the amounts in the kidney, liver, and small intestine are even lower (3). After myocardial damage, H-FABP is released into the intercellular space and appears in the bloodstream (4). The magnitude of the increase in plasma H-FABP has also demonstrated a good correlation with the size of the infarction (5). Myoglobin, another small protein (18 kDa), appears in the plasma within 2–3 h after myocardial infarction and is considered a useful marker in the early detection of AMI (6). Myoglobin lacks specificity because myoglobin released from skeletal muscles cannot be distinguished from that released from the heart. Cardiac troponin I (cTnI) and creatine kinase MB isoenzyme (CK-MB) are more specific for myocardial injury but lack early sensitivity because their blood concentrations do not increase appreciably until 6–8 h after the onset of AMI (7).
The aim of this multicenter study was to compare H-FABP with myoglobin, cTnI, and CK-MB in the early detection of AMI in patients presenting with …
02 Apr 2000
TL;DR: With the Cardiac Reader reliable quantitative results can be easily obtained for both cardiac markers and the system is, therefore, particularly suitable for use in emergency rooms, coronary care units and small hospitals.
Abstract: We present the results of a multicenter evaluation of a new point-of-care system (Cardiac Reader) for the quantitative determination of cardiac troponin T (CARDIAC T Quantitative test) and myoglobin (CARDIAC M test) in whole blood samples The Cardiac Reader is a CCD camera that optically reads the immunochemical test strips The measuring range is 01 to 3 microg/l for CARDIAC T Quantitative and 30 to 700 microg/l for CARDIAC M Both tests are calibrated by the manufacturer The reaction times of the tests are 12 or 8 minutes, respectively Method comparisons were performed with 281 heparinized blood samples from patients with suspected acute coronary syndromes The results obtained with CARDIAC T Quantitative showed a good agreement compared with cardiac troponin T ELISA (r = 089; y = 093x + 002) The method comparison between CARDIAC M and Tina-quant Myoglobin also showed a good agreement between both assays (r = 098; y = 092x + 16) Test lot-to-lot comparisons yielded differences of 2% and 6% for CARDIAC T Quantitative and of 0 to 11% for CARDIAC M The within-run imprecision with blood samples and control materials was acceptable for CARDIAC T Quantitative (CV 10 to 15%) and good for CARDIAC M (CV 5 to 10%) The between-instrument CV was below 7% for CARDIACT Quantitative and below 5% for CARDIAC M The cross-reactivity of CARDIAC T Quantitative with skeletal troponin T was approximately 0003% No significant analytical interference was detected for any of the assays in investigations with biotin (up to 100 microg/l), hemoglobin (up to 0125 mmol/l), hematocrit (26 to 52%), bilirubin (up to 340 micromol/l), triglycerides (up to 50 mmol/l), and 18 standard drugs With the Cardiac Reader reliable quantitative results can be easily obtained for both cardiac markers The system is, therefore, particularly suitable for use in emergency rooms, coronary care units and small hospitals