Showing papers on "Myoglobin published in 1999"

Disruption of myoglobin in mice induces multiple compensatory mechanisms

Abstract: Myoglobin may serve a variety of functions in muscular oxygen supply, such as O2 storage, facilitated O2 diffusion, and myoglobin-mediated oxidative phosphorylation. We studied the functional consequences of a myoglobin deficiency on cardiac function by producing myoglobin-knockout (myo−/−) mice. To genetically inactivate the myoglobin gene, exon 2 encoding the heme binding site was deleted in embryonic stem cells via homologous recombination. Myo−/− mice are viable, fertile, and without any obvious signs of functional limitations. Hemoglobin concentrations were significantly elevated in myo−/− mice. Cardiac function and energetics were analyzed in isolated perfused hearts under resting conditions and during β-adrenergic stimulation with dobutamine. Myo−/− hearts showed no alteration in contractile parameters either under basal conditions or after maximal β-adrenergic stimulation (200 nM dobutamine). Tissue levels of ATP, phosphocreatine (31P-NMR), and myocardial O2 consumption were not altered. However, coronary flow {6.4 ± 1.3 ml⋅min−1⋅g−1 [wild-type (WT)] vs. 8.5 ± 2.4 ml⋅min−1⋅g−1 [myo−/−]} and coronary reserve [17.1 ± 2.1 (WT) vs. 20.8 ± 1.1 (myo−/−) ml⋅min−1⋅g−1 were significantly elevated in myo−/− hearts. Histological examination revealed that capillary density also was increased in myo−/− hearts [3,111 ± 400 mm−2 (WT) vs. 4,140 ± 140 mm−2 (Myo−/−)]. These data demonstrate that disruption of myoglobin results in the activation of multiple compensatory mechanisms that steepen the pO2 gradient and reduce the diffusion path length for O2 between capillary and the mitochondria; this suggests that myoglobin normally is important for the delivery of oxygen.

Skeletal muscle injury induced by eccentric muscle action: muscle proteins as markers of muscle fiber injury.

Abstract: Muscular overuse after high force eccentric muscle action is associated with structural damage of the contractile apparatus that can be observed as Z-line steaming and myofibrillar disruption. Mechanical stress is the major contributing factor for inducing muscle injury, which initiates a cascade of processes resulting in skeletal muscle damage. Disturbances in Ca2+ homeostasis with elevated intracellular [Ca2+] activates the nonlysomal cysteine protease, calpain. Calpain is assumed to play an important role in triggering the response of skeletal muscle protein breakdown, of inflammatory changes, and of regeneration processes in response to eccentric muscle action. The inflammatory response is attributed to changes in hormone and cytokine levels in blood and skeletal muscle. To assess the amount of skeletal muscle damage, plasma CK activity and plasma myoglobin levels have been widely used as markers for muscle injury. As the cytosolic proteins do not necessarily reflect the amount of structural damage, structurally bound proteins such as myosin heavy chains and troponin have been investigated. This paper briefly reviews the cascade of events causing muscle cell injury after unaccustomed eccentric muscle action and the potential of muscle proteins as markers of skeletal muscle damage.

Simultaneous Rapid Measurement of Whole Blood Myoglobin, Creatine Kinase MB, and Cardiac Troponin I by the Triage Cardiac Panel for Detection of Myocardial Infarction

Abstract: This multicenter study evaluated the Biosite Triage® Cardiac Panel as a quantitative, multimarker, whole blood system for the detection of acute myocardial infarction (MI). Optimum cutoffs for the discrimination of acute MI (n = 192 patients, 59 with MI) as determined by ROC curve analyses were as follows: 0.4 μg/L for cardiac troponin I (cTnI); 4.3 μg/L for the creatine kinase MB isoenzyme (CK-MB); and 107 μg/L for myoglobin. The Triage Panel showed the following concordances for detection or rule-out of MI compared with established devices: cTnI >89%; CK-MB >81%; myoglobin >69%. No significant differences were present between methods for the same marker. Diagnostic efficiencies demonstrated comparable sensitivities and specificities for the diagnosis of MI in patients presenting with symptoms compared with the Dade, Beckman, and Behring CK-MB, cTnI, and myoglobin assays; the ratio of sensitivity to specificity for each marker was as follows: cTnI, 98%:100%; CK-MB, 95%:91%; myoglobin, 81%:92%. The areas under the ROC curves for the Biosite myoglobin, CK-MB, and cTnI were 0.818, 0.905, and 0.970, respectively; the areas were significantly different, P <0.05. In patients with skeletal muscle injury and renal disease, the Triage cTnI showed 94% and 100% specificity, respectively. The Triage panel offers clinicians a whole blood, point-of-care analysis of multiple cardiac markers that provides excellent clinical sensitivity and specificity for the detection of acute MI.

Comparative analysis of NMR and NIRS measurements of intracellular PO2 in human skeletal muscle

Abstract: 1H NMR has detected both the deoxygenated proximal histidyl NδH signals of myoglobin (deoxyMb) and deoxygenated Hb (deoxyHb) from human gastrocnemius muscle. Exercising the muscle or pressure cuffi...

Synthesis of sol-gel encapsulated heme proteins with chemical sensing properties

Abstract: Heme proteins such as cytochrome-c (cyt-c), hemoglobin (Hb), and myoglobin (Mb) have been successfully encapsulated in sol-gel derived silica matrices, retaining their spectroscopic properties and chemical function. The thermal stability of cyt-c was significantly improved by immobilization in a porous silica network. Results from optical absorption, resonance Raman, and thermal denaturation studies suggest that biomolecules such as cyt-c design self-specific pores in the silica network according to the size and shape requirements of the biomolecule. Hb and Mb, immobilized using the sol-gel process, bound ligands similar to the proteins in aqueous buffer, and silica-encapsulated manganese myoglobin (MnMb) was a viable detector for nitric oxide (NO).

Measurement of Heme Concentration

Abstract: Heme (iron protoporphyrin IX) is a prosthetic group for a number of hemoproteins in different tissues (e.g., hemoglobin, myoglobin, cytochrome P-450s, mitochondrial cytochromes, catalases, and peroxidases). Mutations in the biosynthetic pathway can affect the synthesis and/or degradation of heme. Several assays are provided in this unit for quantifying heme: a spectrophotometric assay based on the characteristic absorption spectrum of oxidized and reduced form of the hemochrome formed by replacing the nitrogen ligands with pyridine; a fluorescence assay based on removal of the iron by a heated, strong oxalic acid solution to produce fluorescent protoporphyrin; a reversed-phase HPLC assay to measure heme and intermediates in the synthetic pathway; and a radiometric assay to measure newly synthesized heme in tissue culture cells.

Formation and Catalytic Roles of Compound I in the Hydrogen Peroxide-Dependent Oxidations by His64 Myoglobin Mutants

Abstract: A His64 → Asp mutant of sperm whale myoglobin (Mb), H64D Mb, has been prepared to mimic the active site of chloroperoxidase from the marine fungus Caldariomyces fumago, in which distal glutamic aci...

Comparison of femoral blood gases and muscle near-infrared spectroscopy at exercise onset in humans

Abstract: We hypothesized that near-infrared spectroscopy (NIRS) measures of hemoglobin and/or myoglobin O2 saturation (IR-So 2) in the vascular bed of exercising muscle would parallel changes in femoral ven...

Vibrational population relaxation of carbon monoxide in the heme pocket of photolyzed carbonmonoxy myoglobin: Comparison of time-resolved mid-IR absorbance experiments and molecular dynamics simulations

Abstract: The vibrational energy relaxation of carbon monoxide in the heme pocket of sperm whale myoglobin was studied by using molecular dynamics simulation and normal mode analysis methods. Molecular dynamics trajectories of solvated myoglobin were run at 300 K for both the δ- and ɛ-tautomers of the distal His-64. Vibrational population relaxation times of 335 ± 115 ps for the δ-tautomer and 640 ± 185 ps for the ɛ-tautomer were estimated by using the Landau–Teller model. Normal mode analysis was used to identify those protein residues that act as the primary “doorway” modes in the vibrational relaxation of the oscillator. Although the CO relaxation rates in both the ɛ- and δ-tautomers are similar in magnitude, the simulations predict that the vibrational relaxation of the CO is faster in the δ-tautomer with the distal His playing an important role in the energy relaxation mechanism. Time-resolved mid-IR absorbance measurements were performed on photolyzed carbonmonoxy hemoglobin (Hb13CO). From these measurements, a T1 time of 600 ± 150 ps was determined. The simulation and experimental estimates are compared and discussed.

On the significance of hydrogen bonds for the discrimination between CO and O2 by myoglobin

Abstract: Quantum chemical geometry optimisations have been performed on realistic models of the active site of myoglobin using density functional methods. The energy of the hydrogen bond between the distal histidine residue and CO or O2 has been estimated to be 8 kJ/mol and 32 kJ/mol, respectively. This 24 kJ/mol energy difference accounts for most of the discrimination between CO and O2 by myoglobin (about 17 kJ/mol). Thus, steric effects seem to be of minor importance for this discrimination. The Fe—C and C—O vibrational frequencies of CO-myoglobin have also been studied and the results indicate that CO forms hydrogen bonds to either the distal histidine residue or a water molecule during normal conditions. We have made several attempts to optimise structures with the deprotonated nitrogen atom of histidine directed towards CO. However, all such structures lead to unfavourable interactions between the histidine and CO, and to νCO frequencies higher than those observed experimentally.

Tissue release of cardiac markers: from physiology to clinical applications.

Abstract: The early release of cardiac markers is influenced by a variety of factors, the most important influence being their intracellular compartmentation. In contrast to the release of cytosolic proteins, the release of structurally bound proteins requires both a leaky plasma membrane and a dissociation or degradation of the subcellular structure, which is a slower process. Another major impact is the susceptibility to the degradation by cytosolic proteases, such as the calpains. The lysosomes are stable within the first 3-4 hours after onset of ischemia, and, therefore, their enzymes are not involved in the early degradation of structurally bound proteins. Troponin I and troponin T are substrates of μ-calpain. Current experimental as well as clinical results suggest that the molecular mass seems to be of minor importance for the pattern of appearance of myocardial proteins in blood after myocardial infarction. However, within the family of molecules with a certain intracellular compartmentation, the molecular mass is an influence on the appearance in blood, because heavier molecules diffuse at a slower rate, and particularly smaller molecules, such as myoglobin, may enter the vascular system to an even larger extent directly via the microvascular endothelium. The higher the concentration gradient of a marker between the cardiomyocytes and the interstitial space, the faster a parameter will translocate from sarcoplasma to the interstitial space as soon as the plasma membrane permeability is increased. Another influence is local blood and lymphatic flow. Recent experimental studies showed that reperfusion causes a true acceleration of cellular protein leakage by an acute manifestation of plasmalemmal disruptions and not just an enhanced wash out. Marker protein time-courses after myocardial damage are also markedly influenced by their disappearance rate from blood. Most proteins appear to be catabolized in organs with a high metabolic rate, such as liver, pancreas, kidneys, and the reticuloendothelial system. Smaller molecules, such as myoglobin, also pass the glomerular membranes of the kidneys and are reabsorbed and subsequently metabolized in tubular epithelial cells.

Peroxidase Activity of Myoglobin Is Enhanced by Chemical Mutation of Heme-Propionates

Abstract: Peroxidase activity of a myoglobin reconstituted with a chemically modified heme 1 is reported. The heme 1 bearing a total of eight carboxylates bound to the terminal of propionate side chains is incorporated into apomyoglobin from horse heart to obtain a new reconstituted myoglobin, rMb(1), with a unique binding domain structure. The UV−vis, CD, and NMR spectra of rMb(1) are comparable with those of native myoglobin, nMb. The mixing of rMb(1) with hydrogen peroxide yields a peroxidase compound II-like species, rMb(1)-II, since the spectrum of rMb(1)-II is identical with that observed for nMb. Stoichiometric oxidation of several small molecules by rMb(1)-II, demonstrates the significant reactivity. (i) The oxidation of cationic substrate such as [Ru(NH3)6]2+ by rMb(1)-II is faster than that observed for oxoferryl species of nMb, nMb-II. (ii) Anionic substrates such as ferrocyanide are unsuitable for the oxidation by rMb(1)-II. (iii) Oxidations of catechol, hydroquinone, and guaiacol are dramatically enhan...

Myocardial oxygenation in vivo: optical spectroscopy of cytoplasmic myoglobin and mitochondrial cytochromes

Abstract: The oxygenation state of myoglobin and the redox state of cytochromec provide information on the P O 2 in the cytosol and mitochondria, respectively. An optical “window” from ∼540 to 585 nm was fou...

Resonance Raman Investigation of Fe−N−O Structure of Nitrosylheme in Myoglobin and Its Mutants

Abstract: Resonance Raman spectra have been observed for NO adducts of wild-type (WT) sperm whale myoglobin (MbNO) and its H64G, H64L, L29W, V68W, and V68T mutants at neutral and acidic pH. Raman excitation ...

Ultrafast vibrational dynamics of the myoglobin amide i band

Abstract: Mid-infrared transient absorption (“pump−probe”) measurements on the amide I band of myoglobin in D2O and in a glass-forming D2O/glycer(ol-d3) solvent mixture reveal very rapid vibrational energy r...

Myoglobin clearance and removal during continuous venovenous hemofiltration

Abstract: Myoglobin has a relatively high molecular weight of 17,000 Da and is poorly cleared by dialysis (diffusion). However, elimination of myoglobin might be enhanced by an epuration modality based on convection for solute clearances. We present a single case of myoglobin-induced renal failure (peak creatine kinase level: 313,500 IU/l) treated by continuous venovenous hemofiltration (CVVH). Our purpose was to evaluate the efficiency of such a modality using an ultrafiltration rate of 2 to 3 l/h for myoglobin removal and clearance. The hemofilter was a 0.9 m2 polyacrylonitrile (AN69) membrane Multiflow-100 (Hospal-Gambro, St-Leonard, Canada) and the blood flow rate was maintained at 150 ml/min by an AK-10 pump (Hospal-Gambro, St-Leonard, Canada). The ultrafiltration bag was placed 60 cm below the hemofilter and was free of pump control or suction device. Serum myoglobin concentration was 92,000 μg/l at CVVH initiation and dropped to 28,600 μg/l after 18 h of the continuous modality. The mean sieving coefficient for myoglobin was 0.6 during the first 9 h of therapy and this decreased to 0.4 during the following 7 h. Mean clearance of myoglobin was 22 ml/min, decreasing to 14 ml/min during corresponding periods, while the mean ultrafiltration rates were relatively stable at 2,153 ± 148 ml/h and 2,074 ± 85 ml/h, respectively. In contrast to myoglobin, the sieving coefficeint for urea, creatinine, and phosphorus remained stable at 1.0 during the first 16 h of CVVH. More than 700 mg of myoglobin were removed by CVVH during the entire treatment.¶In conclusion, considerable amounts of myoglobin can be removed by an extracorporeal modality allowing important convective fluxes and middle molecule clearances, such as CVVH at a rate of 2 to 3 l/h using an AN69 hemofilter. If myoglobin clearance had been maintained at 22 ml/min, 32 l of serum would have been cleared per day. However, the sieving coefficient of myoglobin decreased over time, probably as a consequence of protein coating and/or blood clotting of the hemofilter. Whereas myoglobin can be removed by CVVH, it remains unknown at this point if such a modality, applied early, can alter or shorten the course of myoglobinuric acute renal failure.

Heme protein dynamics revealed by geminate nitric oxide recombination in mutants of iron and cobalt myoglobin.

Abstract: Nitric oxide myoglobin (MbNO) at 300 K was photodissociated with 405 nm pulses. The NO recombination in several mutants of iron and cobalt myoglobins was investigated at a time resolution of ca. 70 fs. The geminate recombination of NO was nonexponential on sub-nanosecond time scales. For both metals, the change of the detailed structure of the heme pocket (position 68 mutations) caused significant changes in the rates of recombination; however, the metal substitution influenced the recombination much less than did amino acid substitution. The results indicate a primary role of the heme pocket structure in the dynamics, and they suggest that proximal protein relaxation is not the limiting factor in the geminate recombination process. Recombination in cobalt derivatives is somewhat more efficient on the sub-nanosecond time scales than in corresponding iron myoglobins, consistent with other results that show a greater intrinsic reactivity toward the NO of cobalt compared with the iron heme. A comparison of r...

Effect of freezing and microbial growth on myoglobin derivatives of beef.

Abstract: The effect of freezing and bacterial growth on the discoloration of beef was assessed by measuring myoglobin derivatives myoglobin (MB), oxymyoglobin (MBO2), and metmyoglobin (METMB) on the surface...

Near-Infrared Spectroscopic Measurement of Myoglobin Oxygen Saturation in the Presence of Hemoglobin Using Partial Least-Squares Analysis

Abstract: Myoglobin is an important intracellular protein found in cardiac and skeletal muscle. It is involved in the intracellular transport of oxygen from the cell membrane to the mitochondria where oxidative phosphorylation takes place. The optical absorbance characteristics of myoglobin are similar to those of hemoglobin in the nearinfrared spectral region. Distinguishing spectral information of myoglobin from hemoglobin should allow for determination of intracellular oxygen availability in muscle. Partial least-squares analysis is used in this report to determine the oxygen saturation of myoglobin, in the presence of hemoglobin, in vitro. Studies were performed with the use of both transmission and reflectance spectroscopic techniques. Transmission spectra of myoglobin solutions were determined with varying degrees of oxygen saturation achieved by deoxygenating the solution using E. coli. Calibration spectral data sets were developed with the use of varying concentrations of hemoglobin interference, and with varying degrees of myoglobin oxygen saturation. Reflectance spectra were obtained from myoglobin and hemoglobin solutions containing a scattering agent to mimic muscle tissue conditions. Predicted myoglobin saturation values were within 2% of the known saturation values from the use of this analysis. Partial least-squares analysis allows for accurate prediction of myoglobin oxygen saturation in the presence of hemoglobin from either transmission of reflectance near-infrared spectra.