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Biomedical subjects

E Chiancone

Publications and source records attributed to E Chiancone.

At least 109 records · Page 6Linked to original sources
PubMed1983

Binding of terbium to apoferritin: a fluorescence study.

Luminescence measurements show that apoferritin binds three Tb(III) atoms per subunit in accordance with crystallographic evidence. Fe(II) competes with Tb(III) for at least some of the binding sites. This competition may be the molecular basis for the inhibition of iron incorporation into apoferritin brought about by Tb(III). Ca(II), which is generally replaced by Tb(III) in Ca(II) binding proteins, does not compete with the lanthanide for binding to apoferritin.

Animals↗
PubMed1983

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis. The oxidation reaction.

The oxidation by ferricyanide of the dimeric (HbI) and tetrameric (HbII) hemoglobins from the bivalve mollusc Scapharca inaequivalvis has been studied in static and kinetic experiments. Both hemoglobins give rise to hemichromes as stable oxidation products. Oxidation of deoxyHbI yields a hemichrome by a simple bimolecular process. No intermediate Met form can be detected during the reaction even in rapid mixing experiments. The HbI hemichrome undergoes a reversible pH-dependent dissociation into monomers. A simple model has been proposed to account for the linkage between proton binding and subunit dissociation. In the case of tetrameric HbII, oxidation yields an intermediate Met form. Thus, the kinetics of the oxidation reaction are always biphasic; the fast reaction is a bimolecular process and yields the Met derivative. The slow reaction is a monomolecular process and corresponds to the conversion of the Met form into the hemichrome; its rate is independent of the state of ligation of the ferrous protein and decreases with increase of pH. The HbII hemichrome is tetrameric when newly formed; it tends to dissociate into lower molecular weight species with the same optical properties. The rate of dissociation is relatively fast at neutral pH (t 1/2 approximately equal to 12 min) and markedly less at alkaline pH values. The HbI and HbII hemichromes are reduced by dithionite yielding the spectra of the native deoxygenated proteins; in the case of HbII, the tetrameric structure of the native protein is re-acquired.

Animals↗
PubMed1983

Subunit interactions in the dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis.

The dissociation behaviour of oxygenated Scapharea inaequivalvis HbII, the tetrameric hemoglobin component contained in the erythrocytes of this bivalve mollusc, has been compared with that of oxygenated human hemoglobin, HbA. At neutral pH the molluscan protein dissociates reversibly into dimers as does HbA, although dissociation is less marked; moreover the dimer-tetramer association constant is not sensitive to the presence of inorganic phosphates and high salt concentrations. HbII dimers hybridize with HbA dimers in solution, pointing to an overall similarity of the dimer interfaces in these hemoglobins from distantly related species. The gel filtration behaviour of the dimeric hemoglobin component of the erythrocytes. HbI, indicates that at neutral pH the protein has very little tendency to dissociate into monomers even at micromolar concentrations. Hb I was found to contain small amounts (2-4%) of bound carbohydrates.

Animals↗
PubMed1982

Characterization of cation-binding sites on Panulirus interruptus hemocyanin by 43Ca and 23Na NMR.

The binding of Na+ and Ca2+ ions to Panulirus interruptus hemocyanin has been studied by nuclear magnetic resonance. The combined use of 43Ca and 23Na NMR has allowed two different classes of sites to be distinguished. The sites are characterized by a relatively fast chemical exchange, local mobility and binding constants of the order of 10(3)-10(4) M-1 for Ca2+ and Mg2+ and approximately equal to 10(2) M-1 for Na+. These features and the observed pH dependence of the Ca2+ binding are consistent with the protein ligands being carboxylate groups, some of which may occur in clusters.

Binding Sites↗
PubMed1982

Studies on Scapharca hemoglobins. Properties of the dimeric protein reconstituted with Fe- or Co-porphyrin.

A native globin from the dimeric hemoglobin, hemoglobin I, of the mollusc Scapharca inaequivalvis has been obtained with the acid-acetone method. The globin has a lower sedimentation coefficient than the native protein at neutral pH; its reconstitution product with natural heme has the same physicochemical and functional properties as the native protein. proto- and meso-cobalt hemoglobin I have been prepared and characterized. proto-Cobalt hemoglobin I binds oxygen reversibly with a lower affinity and a lower cooperativity than native hemoglobin I; thus, the changes in the functional properties brought about by substitution of iron with cobalt are similar to those observed in human hemoglobin A. The EPR spectra of deoxy-proto-cobalt hemoglobin I and of the photolysis product of oxy-meso-cobalt hemoglobin I indicate that two histidine residues are the apical heme ligands. The broad signal at g = 2.38 in deoxy-proto-cobalt hemoglobin I points to a constrained structure of the heme site in this derivative which results from a distorted coordination of the hindered proximal histidine. A similar structure has been proposed previously for the alpha chains in deoxy-cobalt hemoglobin A.

Apoproteins↗
PubMed1982

Structural heterogeneity and subunit composition of horse ferritins.

Structural, spectroscopic, and immunological properties of horse ferritins extracted from spleen, liver, and heart were studied to test the hypothesis that the different tissue ferritins are hybrids composed of variable proportions of two subunit types. The weight-average molecular weights determined by sedimentation velocity and gel filtration increase from 460 000 for spleen to 480 000 for liver and to 515 000 for heart apoferritin; moreover, the diffusion coefficients prove that each tissue-specific ferritin consists of a population of hybrid molecules. The intrinsic fluorescence and the near-UV circular dichroism (CD) spectra change as a function of the subunit composition of the three ferritins. The fluorescence emission maximum, which occurs at a very low wavelength (315 nm) in horse spleen apoferritin, is shifted to increasingly higher wavelengths in the liver and heart proteins (320 and 325 nm, respectively), indicating that the tryptophan and tyrosine residues become less rigidly immobilized with an increase in the H-subunit content. The tryptophan residues behave as fully solvated in the monomeric subunits at acidic pH values. In accordance with the fluorescence data, the near-UV CD spectra show that the tryptophan environment is highly asymmetric in spleen apoferritin, progressively less so in liver and heart apoferritins, and completely relaxed in the dissociated subunits. Moreover, they show that the environment of tyrosines and phenylalanines differs markedly in the spleen and heart apoproteins. The ferritins studied appear to have immunogenic sites which are specific for the H and L subunits on the basis of enzyme-linked immunoassay and double-diffusion experiments.

Animals↗
PubMed1981

Amphitrite ornata erythrocruorin. II. Molecular controls of function.

In the marine terebellid worm Amphitrite ornata the vascular fluid contains a high molecular weight erythrocruorin, while cells of the coelom contain a monomeric hemoglobin. The structural integrity of the erythrocruorin molecule is known to be dependent on the presence of a minimal concentration of divalent cations (1-3 mM) in the medium. The functional properties of Amphitrite erythrocruorin are also affected by cations. The oxygen affinity tends to increase with increasing cation concentration and the degree of cooperative interactions, expressed in the kinetics and equilibria of ligand binding, goes through a maximum. Maximal Hill coefficients of 3-4 are observed with 50 mM CaCl2, 50 mM MgCl2 or 1 M NaCl in measurements at the physiological pH of 7.75. Only 2 mM CaCl2 is required for maximal cooperativity at pH 8.5. This suggests partial deprotonation of the cation binding site at high pH. It is somewhat unusual that pH effects on cooperativity are reversible, since this is not a common feature of the giant erythrocruorin molecules. The oxygen binding experiments revealed a marked effect of divalent cations of Amphitrite erythrocruorin at high pH and cation concentration. Above pH 8.5, at 50 mM CaCl2 and 12 degrees C, the erythrocruorin will form a polymer upon deoxygenation. This polymerization is readily reversible by bringing the temperature for 12 to 20 degrees C or by oxygenation. Under physiological conditions of pH and cation concentration and at 12 degrees C, the erythrocruorin and the monomeric coelomic hemoglobin require a similar oxygen pressure for half saturation. However, the allosteric regulation of function is absent for the coelomic protein.

Animals↗
PubMed1980

Root effect of Panulirus interruptus hemocyanin.

Panulirus interruptus hemocyanin exhibits a progressive decrease in oxygen affinity and a parallel loss of cooperativity with decrease in pH, resulting in an apparent loss of the oxygen-binding capacity of the protein. For a characterization of this system, oxygen-binding curves have been determined over the complete range of oxygen saturation, applying a special technique which involves high-pressure spectrophotometry. Although the oxygen-binding behavior as a function of pH is complex and cannot be described within the frame of a simple two-state Monod-Wyman-Changeux model, the observed Root effect is clearly related to a progressive stabilization of a low oxygen affinity state of the protein and functional heterogeneity is not apparent.

Animals↗
PubMed1980

Amphitrite ornata erythrocruorin. I. Structural properties and characterization of subunit interactions.

A high molecular weight erythrocruorin (Mr approx. 3 . 10(6)) is found in the vascular system of the marine terebellid worm Amphitrite ornata, while a low molecular weight hemoglobin is contained in the coelomic cells. Polyacrylamide gel electrophoresis indicates that Amphitrite erythrocruorin contains three different types of polypeptide chain, of molecular weight approx. 15,000, whereas the molecular weight per heme group is approx. 20,000. These data suggest that only two of three polypeptide chains may be associated with a heme group. The coelomic hemoglobin, which occurs as a monomer, has an apparent molecular weight of approx. 14,000. The circular dichroism spectra of Amphitrite erythrocruorin and of the coelomic protein reveal marked differences in the heme environment, while the alpha-helical contents are not very different (60% and 70%, respectively). Amphitrite erythrocruorin is unusual in its dissociation behavior. Divalent cations are required for maintaining the quaternary structure. In the pH range 7.75--8.5, when the Ca2+ concentration is reduced below 1 mM, the whole molecule (57 S) dissociates into a number of lower molecular weight species (25, 15, 10 and 3 S) which have been correlated with specific subunit structures by electron microscopy. Whole molecules and 25 S subunits are not in equilibrium with the lower molecular weight species and can be isolated from partially dissociated mixtures. In contrast, the lower molecular weight subunits are themselves in a state of rapid equilibrium which is sensitive to cations, protons and oxygen. Of special interest is the dimerization reaction of the 10 S subunits, which appears to be mediated by Ca2+ and conforms to the predictions of the Cann and Goad theory on ligand mediated equilibria. The dissociation of Amphitrite erythrocruorin is readily reversible when the Ca2+ concentration is increased. The subunits obtained at physiological (7.8) or slightly acid (6.5) pH completely reassemble into whole molecules. Reassembly, however, is only partial when dissociation occurs at high pH. The presence of stable intermediates, such as the 15 S species, may facilitate the reassociation process.

Amino Acids↗
PubMed1979

Sodium and calcium binding to Panulirus interruptus hemocyanin as studied by 23Na nuclear magnetic resonance.

Addition of Panulirus hemocyanin to NaCl solutions produces marked changes in the 23Na relaxation parameters; they show that sodium ions interact with binding sites on the protein and exchange rapidly with the bulk. The observed non-lorentzian lineshapes and the non-exponential decay of the transverse magnetization indicate that non-extreme narrowing conditions apply and give information on the dynamics of the interaction. Panulirus hemocyanin has at least two classes of Na+ binding sites; the binding constant of the more strongly bound sodium ions is in the order of 1 X 10(2) M-1. Competition between Na+ and Ca2+ for protein binding sites is demonstrated by the effect of Ca2+ on the 23Na relaxation parameters. However, only the more strongly bound Na+ are displaced by Ca2+. The number of Ca2+ needed to displace these sodium ions is 3--5 per oxygen binding site. The 23Na relaxation parameters are influenced also by the state of oxygenation of the protein, indicating a linkage between Na+ and oxygen binding. The simplest interpretation of the data is that sodium ions bind more strongly to oxyhemocyanin in agreement with oxygen equilibrium experiments.

Animals↗
PubMed1978

Kinetics of iron reduction of ferritin cores and of a low molecular weight hydroxy-iron polymer.

The kinetics of reduction by dithionite of iron cores in ferritin and of iron cores after removal of the protein shell (isolated cores) has been analysed along with that of a low molecular weight hydroxy-iron polymer. Spectrophotometric measurements have shown that the time course of the reaction approximates a pseudo first-order behaviour. In the case of ferritin cores the pseudo first-order rate constant reaches an asymptotic value which markedly increases when the cores are isolated. In contrast, in the low molecular weight hydroxy-iron polymer, no clear asymptotic value is reached. Thus, the rate of reduction appears to be determined both by the presence of the protein shell and by the crystallite surface area. Kinetic light scattering experiments show that in ferritin and in the isolated cores a rapid drop in molecular weight occurs during the first stages of reduction, suggesting a fragmentation of the iron crystallite.

Ferritins↗