Showing papers on "Myoglobin published in 1987"
TL;DR: The sperm whale myoglobin expressed from the synthetic gene constituted approximately 10% of the total soluble protein as holo-protein, indicating that iron-protoporphyrin IX biosynthesis and prosthetic-group incorporation are not limiting in the high-level expression of this heme protein in E. coli.
Abstract: Sperm whale myoglobin was expressed in Escherichia coli from a totally synthetic gene inserted in the expression vector pUC19. The gene was constructed as 23 overlapping oligonucleotides encoding both strands of the DNA. Gene synthesis provides several advantages over traditional eukaryotic gene-cloning techniques, allowing the incorporation of an efficient ribosome binding site, appropriate initiation and termination sequences, restriction enzyme sites for convenient subcloning and future mutagenesis, and frequently used codons for highly expressed E. coli genes. The sperm whale myoglobin expressed from the synthetic gene constituted approximately 10% of the total soluble protein as holo-protein, indicating that iron-protoporphyrin IX biosynthesis and prosthetic-group incorporation are not limiting in the high-level expression of this heme protein in E. coli. We credit the use of frequently used E. coli codons for the observed high-level expression. The sperm whale myoglobin produced is stable, easily purified to homogeneity, and indistinguishable from commercially available sperm whale myoglobin by optical and magnetic spectroscopic methods.
321 citations
TL;DR: Investigation of the reaction of nitric oxide with ferric heme proteins and model compounds suggests that the presence of an axial water molecule at the ligand binding site of ferric hemoglobin A prevents it from exhibiting significant cooperativity in its reactions with NO.
Abstract: Rates for the reaction of nitric oxide with several ferric heme proteins and model compounds have been measured. The NO combination rates are markedly affected by the presence or absence of distal histidine. Elephant myoglobin in which the E7 distal histidine has been replaced by glutamine reacts with NO 500-1000 times faster than do the native hemoglobins or myoglobins. By contrast, there is not difference in the CO combination rate constants of sperm whale and elephant myoglobins. Studies on ferric model compounds for the R and T states of hemoglobin indicate that their NO combination rate constants are similar to those observed for the combination of CO with the corresponding ferro derivatives. The last observation suggests that the presence of an axial water molecule at the ligand binding site of ferric hemoglobin A prevents it from exhibiting significant cooperativity in its reactions with NO.
243 citations
TL;DR: Muscles with the poorest colour stability, such as Psoas major and Diaphragma medialis had the highest oxidative activities (oxygen consumption rate) and the highest myoglobin autoxidation rates, which does not explain the differences observed in muscle colour stability.
241 citations
TL;DR: It is found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the β subunit are critical for fine-tuning ligand affinity.
Abstract: The geometries of the Fe–O2 and Fe–CO bonds in myoglobin and haemoglobin differ significantly from those in free porphyrin model compounds1–6. It has been suggested that steric hindrance by Val-Ell and His-E7 and a hydrogen bond between His-E7 and oxygen2,4,7 affect the geometry and electronic state of the Fe-ligand bond, and that these interactions may be important in controlling oxygen affinity8. We have produced mutant haemoglobins in E. coli9–11 having Val(67β)E11 replaced by Ala, Met, Leu or Ile and His(58β)E7 by Gin, Val or Gly. We have studied the effect of these mutations on the equilibrium and kinetics of ligand binding. The conformation of the new side chains and their effect on the protein structure have been examined by X-ray crystallography, and the vibrational properties of the Fe–CO bond observed by resonance Raman spectroscopy12. We found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the β subunit are critical for fine-tuning ligand affinity.
155 citations
TL;DR: Serum myoglobin elevation may permit early identification of myocardial infarction, with subsequent verification using CPK-MB determination, allowing appropriate intensive care admission for careful monitoring of these patients.
135 citations
TL;DR: Correlation analysis of levels in each sample indicated skeletal and not cardiac muscle as the source of raised serum protein, and problems arise in distinguishing skeletal from cardiac muscle trauma on the basis of serum enzyme tests following severe muscle exercise.
Abstract: Problems arise in distinguishing skeletal from cardiac muscle trauma on the basis of serum enzyme tests following severe muscle exercise. The contributions of cardiac and skeletal sources have been assessed in eleven marathon runners by measuring pre- and post-race serum levels of cardiac-specific myofibrillar troponin-I together with total creatine kinase, creatine kinase-MB isoenzyme, myoglobin, myofibrillar tropomyosin and C-reactive protein. Total creatine kinase, creatine kinase-MB isoenzyme, tropomyosin and myoglobin were significantly elevated above pre-race levels in all runners between 1 h and 128 h post-race. Neither mean cardiac troponin-I nor C-reactive protein was elevated post-race. Nine out of sixty-three samples fulfilled conventional positive criteria for cardiac muscle damage on the basis of combined creatine kinase and creatine kinase-MB isoenzyme levels. Six runners had one or more positive samples. No samples had levels above twice the upper normal limit for either cardiac troponin-I or C-reactive protein. Correlation analysis of levels in each sample indicated skeletal and not cardiac muscle as the source of raised serum protein.
134 citations
TL;DR: Results demonstrated that, unlike Escherichia coli, H. influenzae may acquire heme from hemoglobin-haptoglobin, and the role of outer membrane proteins in the acquisition of heme is not yet clear.
Abstract: Although Haemophilus influenzae requires heme for growth, the source of heme during invasive infections is not known. We compared heme, lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin as sources of heme for growth in defined media. The minimum concentration of heme permitting unrestricted growth of strain E1a, an H. influenzae type b isolate from cerebrospinal fluid, was 0.02 micrograms/ml. Using molar equivalents of heme as lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin, we determined that myoglobin and hemoglobin permitted unrestricted growth at this concentration. To determine the ability of host defenses to sequester heme from H. influenzae, we used affinity chromatography to purify human haptoglobin and hemopexin, serum proteins which bind hemoglobin and heme. Plate assays revealed that 12 strains of H. influenzae acquired heme from hemoglobin, hemoglobin-haptoglobin, heme-hemopexin, and heme-albumin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of strain E1a grown in heme-replete and heme-restricted conditions revealed a heme-repressible outer membrane protein with an apparent molecular mass of 38 kilodaltons. These results demonstrated that, unlike Escherichia coli, H. influenzae may acquire heme from hemoglobin-haptoglobin. H. influenzae also may acquire heme from hemopexin and albumin, which have not been previously investigated. The role of outer membrane proteins in the acquisition of heme is not yet clear.
123 citations
TL;DR: It is concluded that cardiac mitochondria accept two additive simultaneous flows of oxygen: a flow of dissolved oxygen to cytochrome oxidase and aflow of myoglobin-bound oxygen to a mitochondrial terminus that supports ATP generation by heart cells at physiological ambient oxygen pressure.
Abstract: Myoglobin-mediated oxygen delivery to intracellular mitochondria is demonstrated in cardiac myocytes isolated from the hearts of mature rats. Myocytes are held at high ambient oxygen pressure, 40-340 torr (5-45 kPa); sarcoplasmic myoglobin is fully oxygenated. In this condition oxygen availability does not limit respiratory rate; myoglobin-facilitated diffusion contributes no additional oxygen flux and, since oxygen consumption is measured in steady states, the storage function of myoglobin vanishes. Carbon monoxide, introduced stepwise, displaces oxygen from intracellular oxymyoglobin without altering the optical spectrum of the largely oxidized intracellular mitochondria. A large part, about one-third, of the steady-state oxygen uptake is abolished by carbon monoxide blockade of myoglobin oxygenation. The myoglobin-dependent component of the oxygen uptake decreases linearly with decreasing fraction of intracellular oxymyoglobin, with a slope near unity. Studies using inhibitors of mitochondrial electron transport indicate that myoglobin-delivered oxygen uptake depends on electron flow through the mitochondrial electron transport chain. We conclude that cardiac mitochondria accept two additive simultaneous flows of oxygen: a flow of dissolved oxygen to cytochrome oxidase and a flow of myoglobin-bound oxygen to a mitochondrial terminus. Myoglobin-mediated oxygen delivery supports ATP generation by heart cells at physiological ambient oxygen pressure.
119 citations
TL;DR: The mechanism of action of cytochrome aa3 is the same in red muscle in vivo as in mitochondria in vitro, and an upper bound on the apparent Michaelis constant for maximal VO2 of red muscle is approximately 0.06 Torr.
Abstract: Probability distributions of myoglobin (Mb) saturation and intracellular PO2 were determined with subcellular spatial resolution in dog gracilis muscles during steady-state twitch contraction at 5-...
90 citations
TL;DR: In this article, the authors applied FT-IR spectrometry to the identification of the secondary structure species of a living protein, and obtained the spectra of native myoglobin and albumin with methods using either KBr pellet or film formed on a KBr window from an aqueous solution.
Abstract: FT-IR spectrometry was applied to the identification of the secondary structure species of a living protein. The spectra of native myoglobin and albumin were obtained with methods using either KBr pellet or film formed on a KBr window from an aqueous solution. Pellet preparation of myoglobin and albumin caused the structure to change from α-helix to β-structure. The conformational changes that arise from heat denaturation of myoglobin, albumin, and γ-globulin were observed by the changes in the amide I, II, and III bands. The bands of the 1300, 1260, and 1235 cm−1 regions were respectively assigned to α-helix, disordered, and β-sheet structures. These band positions were substantiated by the spectra of β-lactoglobulin and α-casein. α-Helix structure probably changes to β-structure in the presence of alkali halide, and changes to disordered structure with heat denaturation in phosphate buffer solution. The secondary structure of a protein is further identified by use of the information obtained from the amide I, II, and III bands; the amide III band is especially important. Furthermore, it may be possible to characterize the species of secondary structures of proteins adsorbed on material surfaces.
89 citations
TL;DR: Comparison with Mössbauer spectroscopy indicates that protein dynamics on a time scale faster than 10-7 s is not simply a harmonic process, thus proving that there is no absolute energy minimum for one well defined conformation.
Abstract: The results of X-ray structure analysis of metmyoglobin at 300 K, 185 K, 165 K, 115 K and 80 K are reported. The lattice vectorsa andb decrease linearly with temperature whilec shows non-linearity above 180 K, indicating some type of phase transition. Cooling does change the myoglobin structure but only within the structural distribution as determined by individual 〈x2〉 at room temperature. Two residues showed significant alternative positions for sidechains at higher temperatures while only one position is occupied at low temperatures. In the case of LEU 61 a jump between different positions of the side-chain reduces the potential barrier for the entrance of the O2 molecule to the heme pocket.
TL;DR: Using immunoblots and ELISA and immunoblotting techniques, it was determined that rat heart FABP was localized in the cytosol with no detectable intramitochondrial material and Comparisons between myoglobin and FABp showed thatFABP appeared earlier than myoglobin in development, but myoglobin was more abundant than FABB at birth.
TL;DR: The optical absorption and ligand binding properties of newly reconstituted sperm whale myoglobin were examined systematically and it was found that the orientation of the heme group has no effect on the physiological properties of myoglobin.
TL;DR: The present n.m.r. data indicate that histidines 24 (B5) and 119 (GH1) are hydrogen bonded to each other and, in contrast to neutron diffraction data, show that His24 does not protonate at pH greater than 5.5.
TL;DR: Two strong bands separated by approximately 30 cm-1 for each oxy heme protein subunit indicate that two major protein conformations (structures) that differ substantially in O2 bonding are present.
Abstract: The dioxygen stretch bands in infrared spectra for solutions of oxy species of human hemoglobin A and its separated subunits, human mutant hemoglobin Zurich (beta 63His to Arg), rabbit hemoglobin, lamprey hemoglobin, sperm whale myoglobin, bovine myoglobin, and a sea worm chlorocruorin are examined. Each protein exhibits multiple isotope-sensitive bands between 1160 and 1060 cm-1 for liganded 16O2, 17O2, and 18O2. The O-O stretch bands for each of the mammalian myoglobins and hemoglobins are similar, with frequencies that differ between proteins by only 3-5 cm-1. The spectra for the lamprey and sea worm hemoglobins exhibit greater diversity. For all proteins an O-O stretch band expected to occur near 1125 cm-1 for 16O2 and 17O2, but not 18O2, appears split by approximately 25 cm-1 due to an unidentified perturbation. The spectrum for each dioxygen isotope, if unperturbed, would contain two strong bands for the mammalian myoglobins (1150 and 1120 cm-1) and hemoglobins (1155 and 1125 cm-1). Two strong bands separated by approximately 30 cm-1 for each oxy heme protein subunit indicate that two major protein conformations (structures) that differ substantially in O2 bonding are present. The two dioxygen structures can result from a combination of dynamic distal and proximal effects upon the O2 ligand bound in a bent-end-on stereochemistry.
TL;DR: Hummingbird blood shows a very high unloading efficiency for 02, and the increase in n-value at higher O₂ saturation is important because it prevents arterial desaturation during flight and nocturnal hypothermia.
Abstract: Blood respiratory properties were studied in several species of Brazilian hummingbirds. Three species, Melanotrochilus fuscus, Eupetomena macroura, and Amazilia versicolor, were studied with respect to O₂-Hb equilibrium curves and effect of pH and temperature. O₂ equilibrium of purified hemolyzates and myoglobin solutions from pectoral muscle were studied for Melanotrochilusfuscus. Hematocrit, hemoglobin content, and myoglobin concentration were studied for 11 species. Hematocrits averaged 56.3%; corresponding O₂ capacity was 22.1 vol%, and MCHC 27.0%. Average blood 02 affinity for the three species was 44.0 mmHg at pH 7.40 and 39 C. The Bohr coefficient o was -0.39. The Hill plots were nonlinear and had increasing n-values at higher 02 saturations. The influence of temperature on the O₂-Hb equilibrium of whole blood was moderate, expressed by ΔH values of 7.0-8.0 Kcal mol⁻¹ The data are discussed with focus on the rapid changes in O₂ uptake and body temperature in hummingbirds associated with entry and a...
TL;DR: Molecular dynamics simulations of the tryptophan and heme motions in sperm whale myoglobin were used to calculate the fluorescence intensity and anisotropy decays, and the calculated fluorescence anisotropies exhibited a large subpicosecond decay, corresponding to nondiffusive side-chain motions.
Abstract: The fluorescence of heme proteins is influenced by energy transfer from the excited tryptophan to the heme. Molecular dynamics simulations of the tryptophan and heme motions in sperm whale myoglobin were used to calculate the fluorescence intensity and anisotropy decays. The side chains underwent both small rapid orientational fluctuations and large infrequent transitions between conformations. The predicted motions of the tryptophans and the heme produce large fluctuations in the instantaneous rate of energy transfer, but no stable conformations in which energy transfer is suppressed were found. The calculated fluorescence anisotropies exhibited a large subpicosecond decay, corresponding to nondiffusive side-chain motions. The calculations adequately predict the observed fluorescence decay curve for myoglobin and the total anisotropy decay at 16-ps time resolution. The subnanosecond decays of anisotropy for tryptophan-14 in tuna myoglobin are not reproduced by the calculation.
TL;DR: The results suggest that the tryptophan fluorescence in all met samples are quenched by rapid Forster energy transfer to the heme as predicted from the crystal geometry.
TL;DR: It is concluded that pretranslational mechanisms are important in regulation of myoglobin gene expression in mammalian muscles.
Abstract: We have used blot hybridization techniques and a specific anti-sense RNA probe to determine whether variation in myoglobin gene expression among mammalian striated muscles is attributable to pretranslational regulatory events. We observed that myoglobin mRNA was expressed to approximately 10- and 5-fold greater levels, respectively, in cardiac and soleus (slow-twitch, oxidative, skeletal) muscles of adult rabbits than in tibialis anterior (fast-twitch, glycolytic, skeletal) muscles. Furthermore, when oxidative capacity of tibialis anterior muscles was increased by 21 days of indirect electrical stimulation, a model of exercise conditioning, myoglobin mRNA content increased approximately 15-fold. We conclude that pretranslational mechanisms are important in regulation of myoglobin gene expression in mammalian muscles.
TL;DR: There appears to be a correlation between a structural property of the heme (as inferred from the wavelength of the charge-transfer transition) and a functional property (the CO recombination) of the protein (as inference from the intensity of the transition)
Abstract: The near-infrared charge-transfer transitions at approximately 760 nm in photodissociated hemoglobin and myoglobin display very different time dependences. In photodissociated myoglobin at room temperature the transition has fully relaxed to its deoxymyoglobin value by 10 ns. In photodissociated hemoglobin, the transition is shifted by 6 nm to longer wavelengths at 10 ns. It relaxes about halfway back to the deoxyhemoglobin value by about 100 ns but subsequently changes very slowly out to about 100 microseconds when the signal intensity becomes too small to follow any further. The intensity of this transition, present in only five-coordinate hemes, is found to follow the same time dependence as the wavelength change. Consequently, there appears to be a correlation between a structural property of the heme (as inferred from the wavelength of the charge-transfer transition) and a functional property (the CO recombination) of the protein (as inferred from the intensity of the transition). Possible origins for this correlation are considered.
TL;DR: The similar rate constants and activation energies indicate that the diffusion rate in the protein is determined by the frequency of the conformational changes that open "gates" for the passage of the quencher through the protein.
Abstract: The diffusion of small molecules through the myoglobin structure was studied. It has been shown that the fluorescent Zn-protoporphyrin substitutes easily for the native nonfluorescent Fe-protoporphyrin in myoglobin. The quenching rate of the E-type delayed fluorescence of Zn-protoporphyrin in a substituted myoglobin by the quenchers oxygen and anthraquinonesulfonate was used to measure their diffusion from the ambient solution through the protein to the ligand binding site. The quenching rate constant (at 21 degrees C) for oxygen is kq = (9.6 +/- 0.9) X 10(7) M-1 S-1, only 1 order of magnitude less than that for Zn-hematoporphyrin quenching in aqueous solution. The activation energy in the range between 2 and 40 degrees C is Ea = 6.0 +/- 0.6 kcal/mol. The corresponding data for anthraquinonesulfonate are kq = (2.1 +/- 0.3) X 10(8) M-1 S-1 and Ea = 5.8 +/- 0.6 kcal/mol. Taking into account the statistical factor involved in the oxygen quenching of the Zn-porphyrin triplet, the quenching rates are very similar. The data are discussed in terms of the "gated reaction" theory of Northrup and McCammon. The similar rate constants and activation energies indicate that the diffusion rate in the protein is determined by the frequency of the conformational changes that open "gates" for the passage of the quencher through the protein.
TL;DR: In this article, an isotope effect on the electronic structure of the heme in the low-spin, ferric cyanide complex of sperm whale myoglobin, metMbCN, which can be uniquely attributed to an H-bond between the distal (E7) histidyl imidazole and the bound ligand.
Abstract: The authors demonstrate herein an isotope effect on the electronic structure of the heme in the low-spin, ferric cyanide complex of sperm whale myoglobin, metMbCN, which can be uniquely attributed to an H-bond between the distal (E7) histidyl imidazole and the bound ligand. The perturbation of the heme electronic structure arises directly from the isotope effect of the H bond between the distal His number7 and the bound ligand.
TL;DR: In this article, cyclic voltammetry (CV), single potential step chronoabsorptometry (SPS/CA) and derivative cyclic voltabsorptometrically (DCVA) were used for sperm whale myoglobin reaction at tin-doped indium oxide electrodes.
TL;DR: Temperature-dependent NMR spectral transition of the meso-tetraethylhemin-reconstituted myoglobin was consistent with thermally regulated dynamic free rotation of the hemin in the myoglobin heme pocket.
TL;DR: It is reported previously that the distal(E7) histidine is replaced by glutamine in myoglobin from the shark, Galeorhinus japonicus, and the amino-acid sequence ofMyoglobin from another shark, Heterodontus japanicus, has been determined, indicating that they have a similar geometry in their globin folding.
TL;DR: The results clearly indicate that the rotation of the haem about the alpha-gamma meso axis has little or no effect on the ligand-binding properties of these myoglobins.
Abstract: Ligand-binding kinetics of native and reconstituted sperm-whale myoglobin were studied in relation to haem orientational disorder by rapid kinetic methods. In addition, native yellow-fin-tuna myoglobin with significant amount of haem disorder was also used. The O2 dissociation and association rates were found for the proteins with different degrees of haem disorder, and these results suggest that the isomers are characterized by almost identical kinetic parameters. Rates of CO recombination after photolysis were also identical for the two orientational isomers. The results clearly indicate that the rotation of the haem about the alpha-gamma meso axis has little or no effect on the ligand-binding properties of these myoglobins.
TL;DR: The infrared absorption difference spectra of the amide I/II bands indicate that photolysis and recombination trigger a two-step structural change, which can be explained by the large Gibbs energy of the conformational transition that is necessary to let CO move into the heme pocket.
TL;DR: In this paper, the distribution of soluble iron between three main components (ferritin, haemoglobin plus myoglobin and a low molecular weight fraction) and the pro-oxidant activities of each fraction in heated water-washed muscle systems from beef, pork and mackerel was determined.
TL;DR: The effects of denaturants on the solvent accessibility to tyrosyl residues of apomyoglobin have been examined by means of second-derivative spectroscopy in the near-ultraviolet and it is proved that a refolding occurs in some region of the N-terminal moiety of the molecule.
Abstract: The effects of denaturants on the solvent accessibility to tyrosyl residues of apomyoglobin have been examined by means of second-derivative spectroscopy in the near-ultraviolet. Three apomyoglobins, i.e., sperm whale, horse, and tuna, were selected because of the different distribution of tyrosyl residues in their primary structure. The results are consistent with the occurrence of two independent consecutive events in the guanidine-induced denaturation pattern of apomyoglobin. The first event, which is responsible for the lack of the ability to bind the heme, has been proved to involve conformational changes in both the domains, i.e., segments 1-79 and 80-153, identified in the myoglobin molecule. However, the conformational changes are not of the same type. In fact, the solvent accessibility to tyrosine HC2 is increased probably because of a partial unfolding of the 80-153 domain. Conversely, the solvent accessibility to tyrosine B2 is decreased, thus indicating that a refolding occurs in some region of the N-terminal moiety (1-79 domain) of the molecule.
TL;DR: In the present contribution, the central concepts that have emerged are described and the experimental underpinning will only be sketched in a few places; the details can be found in the references.
Abstract: After a lecture on atoms and molecules a t the Leopoldina Academy, a listener asked: “Can atoms be sick?” Obviously, the answer is no. All atoms of a given element and isotope are exactly alike. Proteins, however, can be sick. The replacement of one amino acid by another one may have disastrous consequences. Biomolecular medicine and pharmacology consequently are important. The entire field is so enormous and the number of possibilities for modification of even a small protein is so incredibly large that tinkering is of very limited value. What we need is a profound understanding of the function and dynamics of biomolecules as one of the essential components for a theoretical medicine. We take the approach of experimental physics to this difficult problem: We select a simple protein (myoglobin), study a simple process (binding of dioxygen and carbon monoxide), construct simple models, and try to fit and explain the data over wide ranges in time, temperature, pressure, and other external conditions. The goal of this approach is the discovery of general concepts and principles that can then also be applied to other systems. The road from such primitive studies to clinical applications is long, but it may well be one that ultimately pays off in a deeper understanding. In the present contribution, I will describe the central concepts that have emerged. The experimental underpinning will only be sketched in a few places; the details can be found in the references.