Showing papers on "Myoglobin published in 1977"

On the nature of the interaction between some anionic polysaccharides and proteins

Abstract: The interactions between myoglobin and bovine serum albumin and the anionic polysaccharides, pectate, alginate and carboxymethyl cellulose, have been studied by differential scanning calorimetry (d.s.c), absorption spectrophotometry and Sephadex chromatography. At pH 6.0 the effect of the polysaccharides on the native proteins is merely to perturb the structure, causing spectral changes in myoglobin and decreased thermal stability in both myoglobin and bovine serum albumin. However, following heat denaturation at this pH much stronger interactions are formed giving rise to stable high molecular weight complexes which inhibit protein-protein aggregation and hence precipitation. The response of these systems to changes in ionic strength and pH suggest that the interactions are primarily electrostatic in nature and increase as the net positive charge on the protein increases.

Serum myoglobin level as diagnostic test in patients with acute myocardial infarction.

Abstract: Serum myoglobin levels were measured in normal subjects and patients by means of a newly developed radioimmunoassay. Myoglobin was identified in all of 135 sera from normal adults and ranged between 6 and 85 ng/ml (mean +/- SE 31 +/- 1.3). Raised myoglobin levels were present in 62 of 64 patients with documented acute myocardial infarction, the mean serum concentration being 528 +/- 76 ng/ml. Serial determinations in 46 patients with acute infarct showed that maximum values usually occurred within 4 hours after admission. In 19 of 42 cases, raised myoglobin levels preceded the rise in creatine kinase (CK) values; in the remaining patients, both serum myoglobin and creatine kinase were increased on admission. Only 2 of an additional 44 patients admitted with chest pain but without subsequent electrocardiographic, enzyme, or technetium-99m stannous pyrophosphate myocardial scintigraphic evidence of acute myocardial infarction had raised myoglobin levels; the mean value for this group was within the normal range (44 +/- 6 ng/ml). Serum myoglobin values also were normal in patients with congestive heart failure without acute myocardial infarction, and in patients after moderate exercise and cardiac catheterisation. Trasient myoglobinaemia appears to be one of the earliest laboratory abnormalities occurring in acute myocardial infarction and, therefore, should prove useful as a diagnostic aid in patients.

Synthetic Models for the Oxygen-Binding Hemoproteins

Abstract: The oxygen binding hemoproteins hemoglobin (Hb), myoglobin (Mb), and cytochrome P-450 are important to the biological transport, storage, and metabolism of oxygen. Nevertheless the nature of the coordinate link between iron and dioxygen in these hemoproteins has not been defined at the atomic level. Furthermore the way in which the glogin proteinheme interaction directs reversible oxygen binding has been obscure. My students have addressed and partially clarified these issues by preparing crystalline iron(II) porphyrin-dioxygen complexes.1–5 These remarkable Mb models which reversibly bind oxygen in solution or in the solid state at ambient temperature have been characterized by Mossbauer1,5, ir spectra4 and X-ray crystallographic analysis.2

A radioimmunoassay for human serum myoglobin: method development and normal values.

Abstract: We describe a radioimmunoassay that will measure both normal and above-normal concentrations of myoglobin in serum. Myoglobin isolated from human pectoralis muscle was purified by (NH4)2SO4 fractionation and Sephadex gel filtration and injected into rabbits to elicit antisera. Myoglobin was radiolabeled by an acylation with [125]-3-(4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester. With the purified myoglobin and antisera, we then developed a radioimmunoassy that involves simultaneous regent addition, a 3.5-h incubation at 37 degrees C, and separation of the antibody-bound fraction by precipitation with polyethylene glycol. Information is given on detection limit, precision, linearity, recovery, and specimen preservation. Cross-reactivity to human hemoglobin is negligible. Finally, we investigated the possible relationship between serum myoglobin concentration and muscle mass.

The reduction by dithionite of Fe(III) myoglobin derivatives with different ligands attached to the iron atom. A study by rapid-wavelength-scanning stopped-flow spectrophotometry.

Abstract: 1. The reductions of a number of sperm whale Fe(III) myoglobin-ligand complexes by sodium dithionite in a phosphate buffer pH 6.4, were investigated by using rapid-wavelength-scanning stopped-flow spectrophotometry. The ligands were azide, cyanide, fluoride, imidazole, thiocyanate and water. 2. The reduction of Fe(III) myoglobin cyanide led to the transient formation of Fe(II) myoglobin cyanide but no intermediate species were observable during the reductions of the other derivatives. The final product of the reaction in all cases was unliganded Fe(II)myoglobin. 3. Invesigation of the effect of dithionite concentration on the rate of reduction indicated that the SO2- radical ion was the active species in reducing the azide, cyanide, fluoride and thiocyanate derivatives. 4. Comparison of the observed rates of reduction at different ligand concentrations with those predicted for a pathway of reduction involving prior dissociation of the ligand, allowed us to estimate the rate of reduction with the ligand in position (outer-sphere reduction). There was a large variation in the relative rates of outer-sphere reduction in the order imidazole greater than CN- greater than SCN- greater than N3- greater than F-. The fluoride derivative was so resistant to outer-sphere reduction that the reaction with SO2- proceeded only by a pathway involving dissociation of F- before reduction. It was calculated that any direct reduction of this complex was at least 100 times slower than that of the azide derivative. 5. The results are discussed in terms of the possible role of the axial ligands in haem proteins and it is suggested that the pathway of the electron to the Fe(III) centre may be via the ppi orbitals of these ligands.

Myoglobin oxidation in ground beef: mechanistic studies

Abstract: Antioxidants, citric acid and ascorbic acid did not affect the initial slow oxidation of myoglobin but extended the time before rapid oxidation began. NaCl increased the initial rate of pigment oxidation. Lipid extracted from freshly ground, lean beef increased the rate of myoglobin oxidation when added to freshly ground lean beef while lipid extracted from ground beef stored for 7 days did not. Added linoleic acid was more effective than trilinolein in promoting myoglobin oxidation. Lipase and lipoxygenase enhanced the rate of pigment oxidation while phospholipase A inhibited it. Oxalate inhibited both lipid and myoglobin oxidation and reversed the pro-oxidant capacity of Fe+2. EDTA also inhibited lipid oxidation but promoted myoglogin oxidation.

Effects of pressure on visible spectra of complexes of myoglobin, hemoglobin, cytochrome c, and horse radish peroxidase.

Abstract: The spectra of the ferric form of most heme proteins [metmyoglobin, methemoglobin, horse radish peroxidase (EC 1.11.1.7), and ferricytochrome c at pH 1.5] are converted from high-spin (open crevice) structure to low-spin (closed crevice) form under pressure. Pressures up to 8000 kg/cm2 (780 MPa) have no effect on the spectra of high-spin ferro- and ferricytochrome c, which have a closed crevice structure at pH 7.0. Spectra of deoxy-ferromyoglobin and deoxy-ferrohemoglobin are reduced in intensity, but pressure does not change the positions of the absorption maxima. Cyanide ion prevents pressure-induced spectral changes in metmyoglobin and methemoglobin up to 8000 kg/cm2. Carbon monoxide (with a high affinity for the ferro heme iron) has a similar effect on ferromyoglobin and ferrohemoglobin. The pressure required to cause spectral changes in the heme proteins falls in the order, cytochrome c (pH 7.0) greater than horse radish peroxidase greater than myoglobin greater than hemoglobin. We have calculated a volume change of --50 cm3/mol associated with the configurational change accompanying the reformation of the iron-methionine bond in cytochrome c at low pH.

Oxidation of myoglobin in vitro mediated by lipid oxidation in microsomal fractions of muscle

Abstract: Enzymic lipid peroxidation of a microsomal fraction prepared from chicken leg muscle led to the oxidation of oxymyoglobin to metmyoglobin when the former was incubated in vitro with the microsomal peroxidation system. Similar oxidation of pigment was observed in the presence of linolenate hydroperoxide. On prolonged incubation of myoglobin with the peroxidizing microsomal fraction, some destruction of the pigment occurred. Incubation with either BHA or a mixture of glutathione and glutathione peroxidase inhibited much of the pigment oxidation.

Prediction and conformation by synthesis of two antigenic sites in human haemoglobin by extrapolation from the known antigenic structure of sperm-whale myoglobin.

Abstract: The complete antigenic structure of sperm-whale myoglobin was previously determined in our laboratory. By structural analogy with myoglobin, two regions in human haemoglobin were predicted to comprise antigenic sites. One region was on the alpha-chain [alpha-(15-23)] and the other on the beta-chain [beta-(16-23)]. These two regions were synthesized, purified and characterized, and their immunochemistry was studied. Each peptide was able specifically to bind considerable amounts of haemoglobin antibodies. In a set of homologous proteins, barring any drastic conformational or electrostatic inductive effects exerted by the substitutions, and allowing for obstruction due to subunit interaction, the determination of the antigenic structure of one protein may serve as a useful starting model for the others.

Nitrosoalkanes as Fe(II) ligands in the hemoglobin and myoglobin complexes formed from nitroalkanes in reducing conditions.

Abstract: Primary and secondary aliphatic nitro compounds, R2CHNO2, react with myoglobin and hemoglobin, in the presence of sodium dithionite, leading to new complexes with Soret peaks respectively at 425 and 421 nm. These complexes are very stable even after disappearance of the starting nitro compounds and do not exchange their exogenous ligand after 10 h under 1 atm (101 325 Pa) CO. They are low-spin hexacoordinated myoglobin or hemoglobin complexes, as shown by the resonance Raman spectrum of the nitromethane-derived human hemoglobin complex which is similar to those of the known hemoglobin complexes with O2, CO, NO and nitrosobenzene. Evidence has been produced to show that the nitro compounds themselves do not bind to the hemoproteins; we propose that among the reduction derivatives produced in situ by dithionite, the corresponding unstable nitroso monomers, whose nitroso group is isoelectronic with dioxygen, are the actual ligands of the 425-nm or 421-nm-absorbing complexes.

Immunological measurements of conformational motility in regions of the myoglobin molecule.

Abstract: The conformational motilities of three regions of the sperm whale myoglobin molecule and of an isolated peptide of myoglobin have been examined by measuring the equilibrium constant for the native equilibrium nonnative transition. The immunological approach of Furie et al. (Furie, B., Schechter, A.N., Sachs D., and Anfinsen, C.B. (1975), J. Mol. Biol.92, 497-506) was used with convenient modifications. Antibodies specific to the nonnative conformations were used in assaying for competition between the radioactively labeled peptide and native myoglobin. Labeling was by 125I iodination of the peptide or its 3-(4-hydroxyphenyl)propionyl derivative, and separation of the immune complex from the free peptide was either by ammonium sulfate precipitation or by centrifugation of the antibodies immobilized on Agarose beads. For the antigenic regions of the sequence (1-55), the measured conformational equilibrium constant was 840 +/- 200 at 22 degrees C; the value for the C-terminal region (132-153) was 280 +/- 120 at 25 degrees C, while that for the region (66-76) adjacent to the heme group was greater than 2.5 x 10(6). Measurements on the isolated peptide (132-153) indicated that 1% of the molecules adopt native-type folding in aqueous solution at 36 degrees C.

Effects of Altitude on Myoglobin and Mitochondrial Protein in Canine Skeletal Muscle

Abstract: Myoglobin and mitochondrial protein contents were measured spectrophotometrically in the sternothyroid muscle of 8 dogs, first in Denver, at a PB of 635 mm Hg, and after a 3-week exposure t

Magnetic resonance studies of the binding of 13C-labeled carbon monoxide to myoglobins and hemoglobins containing modified hemes.

Abstract: The effects of changes in the groups attached to the periphery of the porphyrin ring of the heme of various hemoglobins and myoglobins on the environment experienced by the ligand, carbon monoxide, have been studied by observation of the chemical shift of the bound ^(13)CO. The results indicate that the major interaction between bound ligand and substituents around the porphyrin is that transmitted electronically from substituent to ligand. The nature of the protein environment around the ligand and the interaction between the proximal histidine (F8) and the ligand (through the iron atom) impose differences between subunits of hemoglobin and between myoglobins and hemoglobins which are largely, but not entirely, independent of these substituent effects. To assess the influence of protein structure on the chemical shifts of bound ligand, the shifts of ^(13)CO bound to myoglobin and hemoglobins from a wide range of species have also been measured.

Radioimmunoassay of serum myoglobin in polymyositis and other conditions

Abstract: Radioimmunoassay of serum myoglobin was done in 53 patients with polymyositis syndromes and other conditions. Serum myoglobin values in 33 healthy subjects ranged from 4 to 77 [mean 33.3 +/- 19.8 (SD)] ng/ml. Fifty percent of polymyositis patients had elevated serum myoglobin levels (greater than 80 ng/ml). Serum myoglobin values in polymyositis patients fluctuated more sensitively than CPK and GOT. Combined estimation of myoglobin and CPK offers advantages for the detection of muscle injury and the prediction of disease exacerbation.

A direct measurement of dynamic spin-interconversion rates in the spin-equilibrium protein ferric myoglobin hydroxide.

Abstract: Laser stimulated Raman temperature-jump kinetics were used to measure the spin-interconversion rates K/sub 1/ and k/sub -1/ for horse ferric myoglobin hydroxide. The role of the reported unusual electronic structure for in vivo biological functions was considered especially with respect to the nature of its involvement in electron transfer/storage activity. It was observed that spin-interconversion rate constants greater than 10/sup 7/ S/sup -1/ for this protein are among the fastest measured for any six-coordinate spin-equilibrium compounds. (DDA)

The complete amino acid sequence of the major component myoglobin of dwarf sperm whale (Kogia simus).

Abstract: The complete amino acid sequence of the major component myoglobin from the dwarf sperm whale, Kogia simus, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequenator. Three easily separable peptides were obtained by cleaving the protein at its two methionine residues, and five peptides were obtained from the methyl acetimidated protein by cleavage with trypsin at the four arginine residues. Sequenator analysis of these fragments and the apomyoglobin provided over 80% of the covalent structure of the protein. The remainder of the primary structure was determined by further digestion of the two larger cyanogen bromide fragments with trypsin and staphylococcal protease. To reconfirm many of the substitutions found in this protein, the apomyoglobin was treated with 1,2-cyclohexanedione, and the resulting arginine protected protein was cleaved at its lysine residues with trypsin. This myoglobin differs from that of the sperm whale at 6 positions, and from the other cetacean myoglobins at about 16 positions. The appearance of a histidine residue at position 35 has no precedent in any myoglobin. The substitutions seen at positions 21, 51, and 132 are unique to date for cetacean myoglobins.

Cooperative, low-molecular-weight dimeric myoglobins from the radular muscle of the gastropod mollusc Nassa mutabilis L.

Abstract: Two chromatographically distinct dimeric myoglobins can be isolated from the radular muscles of the sea-snail Nassa mutabilis L. The purification procedure is based on ammonium sulfate fractionation of the centrifuged homogenate followed by CM-cellulose column chromatography and gel filtration through Sephadex G-100. The total recovery, on a heme basis, is about 50%. The two myoglobins show one band by gel electrophoresis and practically identical physical properties. The isoelectric point is at pH 10.2 ± 0.2; the apparent molecular weight is 28000 ± 1500 by gel filtration and is not dependent on the ligand state of the hemes. Ageing of the myoglobin solutions produces a form showing an apparent molecular weight of about 15 000 ± 500 by gel filtration and abnormal spectrophotometric properties. This form is incapable of reassociation. S20, w= 2.0 ± 0.05 S for the native oxy derivative by velocity of sedimentation, S20, w= 1.25 ± 0.005 S for the molecule denatured in sodium dodecylsulfate. Acrylamide gel electrophoresis in sodium dodecylsulfate, in reducing conditions, indicates a molecular weight of about 13000. About 15 000 g protein/mol heme are found by biuret analysis of the isolated globins. Determinations of the absorption coefficients have been carried out by iron analysis and by spectrophotometric measurements on the CN-metmyoglobin and on the pyridine-chromogen derivatives. Oxygen equilibrium curves show that the myoglobin binds cooperatively oxygen with values of the interaction coefficient, n, = 1.5 with no Bohr effect.

Nuclear magnetic resonance studies of substrate-hemoprotein complexes in solution. I. The interaction of xylidine with myoglobin, hemoglobin, and cytochrome P450.

Abstract: 1H nuclear magnetic resonance longitudinal relaxation time (T1) measurements were used to study the interaction of xylidine (2,6-dimethylaniline) with the hemoproteins myoglobin, hemoglobin, and solubilized rat liver microsomal cytochrome P450. Upon addition of various amounts of ferrimyoglobin, ferrihemoglobin, or ferricytochrome P450 to solutions of xylidine, the T1 values for the methyl and phenyl protons of the xylidine molecule decreased markedly. The observed changes showed that xylidine was much more sensitive to addition of cytochrome P450 than to myoglobin or hemoglobin. The relative effects upon the specific moieties of the substrate also differed; whereas myoglobin produced essentially the same effect upon the relaxation rates (T11p-1) of the phenyl and methyl protons of xylidine, hemoglobin and cytochrome P450 produced differential changes in the (T11p-1) values, phenyl > methyl. These results were shown to reflect specific xylidine-ferrihemoprotein complexes. A nonsubstrate, noninteracting internal reference (tetramethylammonium phosphate) was added to each sample as a control both for experimental variation and for effects due to changes in solution viscosity; in no case was a substantial change in the T1 value of the reference observed. Variable-temperature studies confirmed that the residence times for the xylidineferrimyoglobin and xylidine-cytochrome P450 interactions were in the region of fast exchange with respect to the NMR time scale (i.e., τM [unknown] T11M). Formation of the cyano derivative of ferrimyoglobin or ferrihemoglobin changes the paramagnetic spin state from S = 5/2 to S = 1/2, and conversion to the carbonmonoxyferrous derivative results in a diamagnetic species, S = 0. When the ferrihemoproteins were so converted in situ in the presence of xylidine, the T1 values for the xylidine moieties increased. Since the carbonmonoxyferrous derivatives of all three hemoproteins are diamagnetic, the latter type of experiment was performed in all cases to give the control values (1/T10), which allowed calculation of the paramagnetic relaxation rate values, 1/T11p. The 1H T1 results, in conjunction with the value obtained for the dissociation constant of the xylidine-cytochrome P450 complex (KD) = 4.1 x 10-4 M) and the estimated correlation time for the complex, allowed calculation of distances between the heme iron atom and specific portions of the substrate molecule.