Showing papers on "Myoglobin published in 1988"
01 Jan 1988
874 citations
TL;DR: In this time domain the importance of steric features of the protein are less important than the nature of the ligand itself in the geminate recombination process as well as in the relative amounts of the two heme excited states created.
Abstract: On the basis of our time-resolved absorption measurements of hemoglobin (Hb), myoglobin (Mb), and protoheme (PTH), either unligated or ligated with CO, O2, or NO, we propose a description of the photophysics of heme proteins that encompasses their photodissociation, the origin and fate of the observed short-lived transients, and the appearance of the ground-state, unligated heme proteins. Two distinct species are formed upon ligand photodissociation, which occurs in less than 50 fs. We assign these species to excited states of the unligated heme and label them (for the case of hemoglobin) as Hb*I and Hb*II. We suggest that Hb*I is already at least partially domed and has a spin state of at least S = 1. Hb*I decays in 300 fs to the ground-state unligated heme species, which we consider to be S = 2 and at least partially domed. The population of Hb*II varies with the ligand. It is more significant when the ligand is O2 or NO than when the ligand is CO. The similarities of the picosecond and femtosecond bleaching and absorption kinetics of HbCO with those of PTHCO (and of HbNO with those of PTHNO) indicate that in this time domain the importance of steric features of the protein are less important than the nature of the ligand itself in the geminate recombination process as well as in the relative amounts of the two heme excited states created. It is suggested that the quantum yield of ligand photodissociation is unity whether the ligand is O2, NO, or CO. The low yield of photodissociated heme-O2 or heme-NO compounds as measured on the microsecond time scale is thus attributed to a fast (2.5 ps) recombination of O2 or NO with Hb*II. We discuss geminate recombination measurements of cyanomet hybrid hemoglobins with NO and consider these results in terms of alpha and beta subunit heterogeneity. The first picosecond transient absorption spectra of cyanomet-CO hybrid hemoglobins are presented and are compared with the spectra of other heme compounds. The superimposability of the transient spectra on the equilibrium spectra of heme compounds that exhibit minimal or no cooperativity is noted and is compared with the case of cooperative systems where the transient spectra are distorted with respect to the equilibrium spectra. This distortion is interpreted in terms of an interaction of a domed heme with the F helix.
351 citations
TL;DR: Investigation of the effects of the iron chelator deferoxamine in two experimental models of pigment-induced acute renal failure found it attenuated the fall in glomerular filtration rate after ischemia and hemoglobin infusion, and also associated with significant lipid peroxidation.
Abstract: In ischemic acute renal failure oxygen free radicals may mediate injury. In addition, iron appears to play a critical role in hydroxyl radical formation and lipid peroxidation during reperfusion of...
297 citations
TL;DR: Structural differences in the oxygen binding pockets shown by X-ray crystallographic studies account for the functional differences of these proteins.
Abstract: The distal E7 histidine in vertebrate myoglobins and haemoglobins has been strongly conserved during evolution and is thought to be important in fine-tuning the ligand affinities of these proteins. A hydrogen bond between the N epsilon proton of the distal histidine and the second oxygen atom may stabilize O2 bound to the haem iron. The proximity of the imidazole side chain to the sixth coordination position, which is required for efficient hydrogen bonding, has been postulated to inhibit sterically the binding of CO and alkyl isocyanides. To test these ideas, engineered mutants of sperm whale myoglobin and the alpha- and beta-subunits of human haemoglobin were prepared in which E7 histidine was replaced by glycine. Removal of the distal imidazole in myoglobin and the alpha-subunits of intact, R-state haemoglobin caused significant changes in the affinity for oxygen, carbon monoxide and methyl isocyanide; in contrast, the His-E7 to Gly substitution produced little or no effect on the rates and extents of O2, CO and methyl isocyanide binding to beta-chains within R-state haemoglobin. In the beta-subunit the distal histidine seems to be less significant in regulating the binding of ligands to the haem iron in the high affinity quaternary conformation. Structural differences in the oxygen binding pockets shown by X-ray crystallographic studies account for the functional differences of these proteins.
262 citations
TL;DR: Chemically modified electrodes exhibiting electrocatalytic response toward myoglobin and hemoglobin were constructed by adsorbing the phenothiazine mediator titrants methylene blue and thionine onto spectroscopic graphite.
Abstract: Chemically modified electrodes (CMEs) exhibiting electrocatalytic response toward myoglobin and hemoglobin were constructed by adsorbing the phenothiazine mediator titrants methylene blue and thionine onto spectroscopic graphite. These CMEs, which were prepared by a rapid (60 s) and reproducible (3.2% relative standard deviation) dip-coating procedure, permitted the hemoprotein electroreduction to take place at the reduction potential of the mediator molecule
207 citations
169 citations
TL;DR: Replacement of the prosthetic group of sperm whale myoglobin with zinc protoporphyrin IX prevents H2O2-induced dimerization even when intact horse metmyoglobin is present in the incubation, which suggests that the tyrosine radicals required for the dimerized reaction are generated by intra- rather than intermolecular electron transfer to the ferryl heme.
163 citations
TL;DR: The separation of a protein mixture by charged ultrafiltration membranes was studied and it was found that rejection of the protein was low, while it was high at the pH level which gave the protein the same sign of charge as that of the membrane.
155 citations
TL;DR: Using the isolated perfused rat hindlimb and the fluorocarbon-transfused rat, the optical characteristics of the rat skeletal muscle in the near-infrared region are examined to determine the ratio of absorption coefficients at 700, 730, and 805 nm of oxy- and deoxy-hemoglobins of blood in the thigh muscle.
Abstract: Using the isolated perfused rat hindlimb and the fluorocarbon-transfused rat, we have examined the optical characteristics of the rat skeletal muscle in the near-infrared region. The total contribution of myoglobin and cytochromes to the overall absorbance change was less than 10%. Analyzing transmitted light at 700, 730, and 805 nm, we found linear relationships between the absorbance and the hemoglobin concentrations at hematocrit values from 15 to 50% in the inflowing perfusate. Based on the relationship, we determined the ratio of absorption coefficients at 700, 730, and 805 nm of oxy- and deoxy-hemoglobins of blood in the thigh muscle. The values in thigh muscle were significantly smaller than those in hemoglobin solutions for deoxygenated blood. On the other hand, the values in thigh muscle were larger than those in hemoglobin solutions for oxygenated blood. Solving simultaneous equations by the use of these absorption coefficients, we calculated the changes in the contents of oxy-, deoxy-, and total hemoglobins in the anesthetized rat hindlimb under various conditions. The oxygen saturation of blood determined by our optical method in the thigh muscle was very close to that in the vena cava measured directly with a gas analyzer.
153 citations
TL;DR: It is indicated that antigen processing is a major factor, in addition to MHC binding and T cell repertoire, that determines Ir gene responsiveness and epitope immunodominance and processing of native myoglobin may influence the apparent specificity of the T cell response.
Abstract: Two lines of evidence in the current study indicate that antigen processing is a major factor, in addition to MHC binding and T cell repertoire, that determines Ir gene responsiveness and epitope immunodominance. First, immunization with synthetic peptides of myoglobin sequences revealed new reactivities that had not appeared after priming with native myoglobin. For example, B10.S mice (H-2S) immune to equine myoglobin predominantly responded to peptide 102-118, whereas there was little, if any, response to this peptide in B10.BR (H-2k) mice immunized with native equine myoglobin. However, after immunization with the 102-118 peptide, both strains responded to the peptide. After in vitro restimulation, B10.BR T cells responded as well as B10.S T cells. Similarly, some individual 102-118-specific T cell clones from mice of both haplotypes showed similar dose responses and fine specificity patterns. Thus, low responsiveness to this site is due neither to a hole in the repertoire nor to a failure to bind to the appropriate MHC molecule. An alternative explanation was suggested by the observation that, whereas B10.S T cells from peptide 102-118-immune mice responded almost as well to whole myoglobin as to the peptide, the B10.BR T cells from peptide immune mice, while responding well to peptide, were poorly stimulated by whole myoglobin. Thus, the product of natural processing of equine myoglobin probably has hindering structures in the regions flanking the core epitope 102-118 that interfere with presentation by I-Ak but not I-AS. The second line of evidence that processing of native myoglobin may influence the apparent specificity of the T cell response was obtained using the I-Ad-restricted sperm whale myoglobin 102-118-specific clone 9.27. This clone discriminated readily between whole sperm whale myoglobin and equine myoglobin, but it did not distinguish between peptides corresponding to 102-118 of the sperm whale and equine sequences. This distinction between equine peptide and native equine myoglobin could be overcome by artificial "processing" of equine myoglobin with cyanogen bromide. In both sets of experiments, F1 APCs that present the same epitope well to T cells of another haplotype failed to overcome the defect, which was therefore not due to the availability of different processed cleavage fragments in APC of different haplotypes, as would be expected if there were MHC-linked processing. Thus, the differential responses to peptides versus native molecule for both I-Ad- and I-Ak-restricted clones appeared to depend on the restricting molecule used.(ABSTRACT TRUNCATED AT 400 WORDS)
129 citations
01 Jan 1988
TL;DR: It is concluded that intact oxymyoglobin or metmyoglobin molecules do not react with H2O2 to form .OH detectable by deoxyribose, but that H2 O2 eventually leads to release of iron ions from the proteins, which can react toForm .OH outside the protein or close to its surface.
Abstract: Incubation of horse-heart oxymyoglobin or metmyoglobin with excess H2O2 causes formation of myoglobin(IV), followed by haem degradation. At the time when haem degradation is observed, hydroxyl radicals (.OH) can be detected in the reaction mixture by their ability to degrade the sugar deoxyribose. Detection of hydroxyl radicals can be decreased by transferrin or by OH scavengers (mannitol, arginine, phenylalanine) but not by urea. Neither transferrin nor any of these scavengers inhibit the haem degradation. It is concluded that intact oxymyoglobin or metmyoglobin molecules do not react with H2O2 to form OH detectable by deoxyribose, but that H2O2 eventually leads to release of iron ions from the proteins. These released iron ions can react to form OH outside the protein or close to its surface. Salicylate and the iron chelator desferrioxamine stabilize myoglobin and prevent haem degradation. The biological importance of OH generated using iron ions released from myoglobin by H2O2 is discussed in relation ...
TL;DR: Five monoclonal antibodies against sperm whale myoglobin have been used to investigate the physical state of the antigen adsorbed onto a polydimethylsiloxane surface and the specific loss of certain antigenic determinants on the adsorbing myoglobin indicates a nonrandom adsorption of the myoglobin molecules.
TL;DR: In this paper, it was shown that myoglobin has a globin moiety that can protect the FeO 2 center from easy access of a water molecule and its conjugate anionic species.
TL;DR: Flash photolysis data provide evidence for a larger distal pocket and a smaller enthalpy barrier for [GlyE7]MbCO as compared with wild-type MbCO.
Abstract: Low-temperature flash photolysis with IR and visible spectroscopy was used to probe the influence of the distal histidine His-64(E7) of sperm-whale myoglobin (Mb) on the orientation of bound carbon monoxide (CO) and on the kinetics of CO rebinding. The synthesis and high-level expression of a sperm-whale myoglobin gene in Escherichia coli permits the efficient substitution of the distal histidine through site-directed mutagenesis. Substitution of His-E7 with glycine [GlyE7]Mb bound with CO (CO[GlyE7]Mb) results in one broad bound-CO IR stretch band, v(C-O), centered at 1973 cm-1 at 10 K, in contrast to three distinct bands for native and synthetic wild-type MbCO at 1966, 1945, and 1929 cm-1. After flash photolysis at 10 K, the unbound state of CO[GlyE7]Mb exhibits two CO stretch bands, whereas MbCO has three. Fourier transform IR spectroscopy measurements of the linear dichroism after photoselective flash photolysis of CO bound to [GlyE7]Mb at 10 K reveals the bound CO to be oriented at an angle of alpha = 20 degrees +/- 2 degrees with respect to the heme normal. Flash photolysis data from 10 to 300 K provide evidence for a larger distal pocket and a smaller enthalpy barrier (by approximately 4 kJ/mol) for [GlyE7]MbCO as compared with wild-type MbCO. These results reinforce the notion that the dominant control of the binding step at the heme iron comes from the proximal side through the protein structure.
TL;DR: A 96 picosecond dynamics trajectory of myoglobin with five xenon-probe ligands in internal cavities is examined to study the effect of protein motions on ligand motion and internal cavity fluctuations.
TL;DR: There is a universal, temperature-independent, correlation between spectral shift and survival probability in the rebinding kinetics, and the same quantitative model which accounts for rebinding accounts semiquantitatively for the temporal shift in the peak.
Abstract: The temporal shift in the near-IR absorption peak of myoglobin (Mb) following flash photolysis of MbCO at cryogenic temperatures appears to be due largely to an inhomogeneous reactive process rather than to relaxation. This conclusion, which follows from a new analysis of the experimental data, is based on the following three points: First, at very low temperatures (60 K) a transient line-narrowing effect can be detected. Second, there is a universal, temperature-independent, correlation between spectral shift and survival probability in the rebinding kinetics, and third, the same quantitative model which accounts for rebinding accounts semiquantitatively for the temporal shift in the peak. A fit to the model indicates that the inhomogeneous broadening of the near-IR peak in myoglobin is 15-20% of the total width. The same rebinding process which governs the loss of intensity of this peak is therefore most likely responsible for the shift in its center wavelength.
TL;DR: The differences between the CO-, O2-, and NO-binding parameters for R and T state hemoglobin appear to be due to a decrease in the geminate reactivity of the heme iron atom, with little or no change in the accessibility of the distal pocket.
TL;DR: The kinetic results of the reconstitution reaction of ferric cyanomyoglobin from apomyoglobin and hemin dicyanide showed that the polypeptide chain is entirely folded before the completion of three-dimensional structure of the heme pocket.
Abstract: The reconstitution reaction of ferric cyanomyoglobin from apomyoglobin and hemin dicyanide was investigated with a stopped-flow apparatus by the use of five kinds of probes; (a) Soret absorption, (b) fluorescence quenching of tryptophan, (c) far-ultraviolet CD, (d) near-ultraviolet CD, and (e) Soret CD. After mixing of apomyoglobulin with equimolar amounts of hemin dicyanide, the Soret absorption band was shifted to longer wavelengths within 10 ms. The shifted band kept its shape for a few seconds, and then gradually shifted to shorter wavelengths. A rate constant of the slow reaction was 1.1 x 10(-2) s-1. Time courses of fluorescence quenching followed a second-order reaction with a rate constant of 9 x 10(7) M-1 s-1. Far-ultraviolet CD recovered to the level of native state within the response time of an apparatus (= 64 ms). Near-ultraviolet CD and Soret CD changed with first-order rate constants of 5-30 s-1 and 5 x 10(-3) s-1 respectively. On the basis of the kinetic results we propose the following reconstitution pathway of myoglobin. Apomyoglobin has essentially a highly folded structure similar to myoglobin, but there are some differences in the secondary structure between them. In the first step, heme enters the pocket-like site of apomyoglobin and interacts with surrounding hydrophobic residues in the pocket, and then the interaction may give a complete ordered structure to the protein. Second, the tertiary structure of the heme pocket is partly constructed. Third, the iron-proximal His bond occurs, followed by the attainment of the final conformation. This sequence of the events shows that the polypeptide chain is entirely folded before the completion of three-dimensional structure of the heme pocket. The reconstitution pathway is fairly different from that of the alpha subunit of hemoglobin reported by Leutzinger and Beychok [Proc. Natl Acad. Sci. USA (1981) 78, 780-784], which described how a drastic recovery in helicity was observed on the heme-binding, and that the recovery is introduced by the formation of the heme pocket structure. The difference in the results found for the alpha subunit and myoglobin suggests a difference in conformation: in apomyoglobin most of the helices are arranged and folded around a helix core to form a compact structure as a whole, while in apo-alpha subunit some helices are not folded around the helix core. Helix D, which is absent in the alpha subunit, may play an important role in folding of the helices.
TL;DR: The optical absorption spectra of sperm whale deoxy‐, oxy‐, and carbonmonoxymyoglobin in the temperature range 300–20 K and in 65% glycerol or ethylene glycol–water mixtures are reported.
Abstract: Synopsis We report the optical absorption spectra of sperm whale deoxy-, oxy-, and carbonmonoxymyoglobin in the temperature range 300-20 K and in &5'% glycerol or ethylene glycol-water mixtures. By lowering the temperature, all bands exhibit half-width narrowing and peak frequency shift; moreover, the near-ir bands of deoxymyoglobin show a marked increase of the integrated intensities. Opposed to what has already been reported for human hemoglobin, the temperature dependence of the first moment of the investigated bands does not follow the behavior predicted by the harmonic Franck-Condon approximation and is sizably affected by the solvent composition; this solvent effect is larger in liganded than in nonligmded myoglobin. However, for all the observed bands the behavior of the second moment can be quite well rationalized in terms of the harmonic Ranck-Condon approximation and is not dependent on solvent composition. On the basis of these data we put forward some suggestions concerning the structural and dynamic properties of the heme pocket in myoglobin and their dependence upon solvent composition. We also discuss the different behaviok of myoglobin and hemoglobin in terms of the different heme pocket structures and deformabilities of the two proteins.
TL;DR: Results from four experiments indicated that reduction could be described by a first‐order, irreversible reaction having an average rate constant of 0.0164 min‐1 (22 degrees C) and the unexpectedly low value of DAPP found in intact fibres might be due to the binding of myoglobin to relatively immobile sites in myoplasm.
Abstract: 1. Experiments were carried out on intact, single skeletal muscle fibres from frog in order to estimate the apparent diffusion constant of myoglobin (denoted DAPP) in the myoplasm of living muscle cells. An optical technique was employed to measure myoglobin concentration along the fibre axis following injection of metmyoglobin (denoted metMb) at a point source. The concentration profiles were fitted by the one-dimensional diffusion equation to give estimates of DAPP. The method relied on the fact that myoglobin is normally absent from these frog fibres, thus permitting resolution of the myoglobin-related absorbance above the intrinsic absorbance of the fibre. 2. One complication in the method was that metMb became significantly reduced to oxymyoglobin (denoted MbO2) during the elapsed time before measurement of the concentration profile. The rate of reduction was evaluated by fitting myoglobin-related absorbance spectra, measured at different times following injection of metMb, with in vitro absorbance spectra of metMb and MbO2. Results from four experiments indicated that reduction could be described by a first-order, irreversible reaction having an average rate constant of 0.0164 min-1 (22 degrees C). The effect of reduction on the fitting of DAPP was taken into account. 3. DAPP was determined under three fibre conditions: (1) long sarcomere spacing (3.6-3.8 microns) at 16 degrees C, (2) long sarcomere spacing at 22 degrees C, and (3) normal sarcomere spacing (2.4-2.7 microns) at 22 degrees C. The average values for DAPP under these conditions were: (1) 0.12 (n = 5); (2) 0.17 (n = 5); and (3) 0.15 (n = 7) x 10(-6) cm2 s-1. The average value at 22 degrees C, 0.16 x 10(-6) cm2 s-1, is about 4 times smaller than values for myoglobin diffusivity at 20 degrees C commonly assumed in models of facilitated transport of oxygen by myoglobin. 4. In order to test the possibility that the unexpectedly low value of DAPP found in intact fibres might be due to the binding of myoglobin to relatively immobile sites in myoplasm, experiments were carried out in a cut-fibre preparation using a technique described by Maylie, Irving, Sizto & Chandler (1987 b) for determining the diffusion constants and degree of myoplasmic binding of absorbance dyes. Values for DAPP and the factor (denoted 1 + beta) by which the total myoglobin concentration exceeded the free myoglobin concentration were obtained by fitting the absorbance data by solutions of the one-dimensional diffusion equation.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: In this paper, a detailed physical picture of protein dynamics has been obtained for myoglobin using Mossbauer spectroscopy on 57Fe with a characteristic time faster than 100 ns for protein dynamics with no well defined energy minimum.
Abstract: Mossbauer spectroscopy on57Fe allows the study of dynamics with a characteristic time faster 100 ns. For myoglobin a detailed physical picture of protein dynamics has been obtained. A myoglobin molecule has no well defined energy minimum. X-ray structure analysis yields only an average conformation. At low temperatures the molecules are trapped in slightly different structures called conformational substates. At higher temperatures a Brownian type of oscillation of molecular segments in restricted space occurs. RSMR technique allows an estimation of the characteristic size of these segments which are in myoglobin well below 30 A and larger than 6 A. A determination of the quasielastic absorption with high accuracy yields the energy distribution of the conformational substates. As further examples bacteriorhodopsin and a model compound for membranes are discussed.
TL;DR: In this paper, the effectiveness of myoglobin and its derivatives as photosensitizers in singlet oxygen (1O2) generation was determined by electron paramagnetic resonance (EPR) spectroscopy using a spin trapping technique.
Abstract: The effectiveness of myoglobin and its derivatives as photosensitizers in singlet oxygen (1O2) generation was determined by electron paramagnetic resonance (EPR) spectroscopy using a spin trapping technique. A stable nitroxide radical adduct, 2,2,6,6,-tetramethyl-4-piperidone-N-oxyl (TAN), was formed and detected by EPR. Native myoglobin and apomyoglobin exhibited no photosensitizing function, whereas hematin appeared to be a weak sensitizer. Protoporphyrin IX ring showed strong photosensitizing activity. A 1O2 quencher, N,N N′N′-tetramethylethylene diamine (TMEDA) and the combination of TMEDA and a free radical scavenger, butylated hydroxyanisole (BHA) drastically reduced the EPR signal of TAN when protoporphyrin IX solution was illuminated indicating that 1O2 was produced using protoporphyrin IX ring as a sensitizer.
TL;DR: It is proposed that serum myoglobin is a reliable measure of myocardial necrosis and serves to detect a hitherto undefined population of small-size acuteMyocardial infarction, with its attendant clinical and prognostic implications.
Abstract: • Serum myoglobin levels were studied in 178 consecutive patients admitted for chest pain due to ischemic cardiac injury. Serum myoglobin level was compared with the clinical condition, electrocardiographic changes, and serum creatine kinase levels. Elevated serum myoglobin concentration was present in all patients with acute myocardial infarction, as defined by World Health Organization, Geneva, criteria, and, in addition, in about 50% of patients with so-called acute coronary insufficiency. On this basis we could define two different groups of patients with acute coronary insufficiency: cases exhibiting elevated serum myoglobin levels (group 1) and those with normal levels (group 2). In group 1 although creatine kinase levels were in the normal range, they were significantly higher than in group 2. Four patients from group 1 developed heart failure and another a typical acute myocardial infarction during hospitalization, whereas no patients of group 2 had such complications. In patients with acute myocardial infarction, the elevation of serum myoglobin preceded that of creatine kinase in most cases. Myoglobin release appears to be related to infarct size, the highest levels were found in extensive myocardial infarction and less marked elevations in cases of subendocardial infarction and in half of the cases with acute coronary insufficiency. It is proposed that serum myoglobin is a reliable measure of myocardial necrosis and serves to detect a hitherto undefined population of small-size acute myocardial infarction, with its attendant clinical and prognostic implications. ( Arch Intern Med 1988;148:1762-1765)
TL;DR: Time-resolved circular dichroism and absorption spectroscopy are used to follow the photolysis reaction of (carbonmonoxy)myoglobin and suggest the existence of an intermediate found after ligand loss from MbCO that is similar in structure to the final Mb product.
TL;DR: In this paper, the relative proportions of α-helix, β-structure, and random coil were estimated by simulating a mixed CD spectrum of reference spectra of the corresponding structures to the experimentally obtained CD spectrum.
TL;DR: Results indicate that iron OEP serves as a prosthetic group for myoglobin with normal function, despite the significant structural and electronic difference between OEP and protoporphyrin.
TL;DR: Data indicate that solvent properties influence the haem pocket stereodynamics in myoglobin; moreover, the different behaviour between myoglobin and haemoglobin suggests that the process should involve the surfaces that are buried in the ha Hemoglobin tetramer and exposed to the solvent in myemia, and/or the different protein compressibility.
TL;DR: A graphical analysis of model results is used to illustrate interactions among the main determinants of O2 transport between hemoglobin (Hb) and cytochrome.
Abstract: This paper relates spectroscopic determinations of myoglobin (Mb) saturation and PO2 in individual myocytes (Gayeski and Honig, 1986) to a mathematical model of O2 transport (Federspiel, 1983). A graphical analysis of model results is used to illustrate interactions among the main determinants of O2 transport between hemoglobin (Hb) and cytochrome. The results of the analysis are summarized in a plot called the O2 release curve. The results are qualitatively applicable to normal and pathophysiology, and to Mb-free tissues.