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D Verzili

Publications and source records attributed to D Verzili.

26 records · Page 2Linked to original sources
PubMed1984

Kinetics of ligand binding and quaternary conformational change in the homodimeric hemoglobin from Scapharca inaequivalvis.

The kinetics of the reaction with oxygen and carbon monoxide of the homodimeric hemoglobin from the bivalve mollusc Scapharca inaequivalvis has been extensively investigated by flash and dye-laser photolysis, temperature jump relaxation, and stopped flow methods. The results indicate that cooperativity in ligand binding, already observed for oxygen at equilibrium, finds its kinetic counterpart in a large decrease of the oxygen dissociation velocity in the second step of the binding reaction. In the case of carbon monoxide, cooperativity is clearly evident in the increase of the combination velocity constant as the reaction proceeds. Therefore, the ligand-binding kinetics of this dimeric hemoglobin shows the characteristic features of the corresponding reactions of tetrameric hemoglobins. Analysis of the data in terms of the allosteric model proposed by Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) has shown that the values of the allosteric parameters cannot be fixed uniquely for a dimeric hemoglobin. The rapid changes in absorbance observed at the isosbestic points of unliganded and liganded hemoglobin following laser photolysis provided a value of 7 X 10(4) S-1 at 20 degrees C for the rate of the ligand-free quarternary conformational change, postulated on the basis of cooperative ligand binding. Comparison of the rapid absorbance changes observed during ligand rebinding in this hemoglobin with those observed in tuna hemoglobin indicate that, at full photolysis, binding to the T state is followed by further binding and conversion to the liganded R state; at partial photolysis, population of the liganded T state occurs immediately and is followed by a decay to the liganded R state upon further ligand binding. These new results, in conjunction with previous equilibrium data on the same system, show unequivocally that the presence of two different types of chain is not an absolute prerequisite for cooperativity in hemoglobins, contrary to currently accepted ideas.

Animals↗
PubMed1984

Assembly of erythrocruorin from the earthworm Octolasium complanatum.

The spontaneous assembly of the earthworm erythrocruorin molecule (60 S) from its 1/12 subunits (10 S) obtained by alkaline dissociation is a long debated problem, since the 60 S to 10 S dissociation step has been regarded as essentially irreversible or as only partially reversible when freshly dissociated solutions are used. Erythrocruorin from the earthworm Octolasium complanatum has been reassembled from its 10 S subunits. "Age" of the subunits, pH, and divalent cation concentration are the factors that influence the assembly reaction. Of primary importance is the age of the subunits, i.e. their exposure time to the alkaline dissociating pH. Parallel sedimentation velocity and sodium dodecyl sulfate/polyacrylamide gel electrophoresis experiments on the dissociated and reassembled solutions indicate that two processes take place at alkaline pH values: disulfide exchange and limited proteolysis. These processes, whose relative importance differs in the various preparations, might be responsible for the loss of reassociating capacity of the 10 S subunits. With freshly dissociated subunits, reassembly up to 80% may be achieved at pH 6.2 to 6.5 in the absence of divalent cations; the presence of 25 to 50 mM-Ca2+ renders the reaction essentially pH-independent in the range 6.2 to 8. The effect of Ca2+ is discussed in the light of the presence of structure-stabilizing binding sites for divalent cations at the 10 S intersubunit's contact regions.

Animals↗
PubMed1983

Thermodynamic properties of oxygen equilibria of dimeric and tetrameric hemoglobins from Scapharca inaequivalvis.

Oxygen equilibrium curves of the dimeric and tetrameric hemoglobin components of Scapharca inaequivalvis were determined at several temperatures. The oxygen equilibrium curves were analyzed by the two-step and four-step oxygen equilibrium schemes of Adair for the dimeric and tetrameric hemoglobins, respectively. The enthalpy and entropy changes for each oxygenation step were determined by the temperature dependences of the Adair constants using van't Hoff equations. Neither dimeric nor tetrameric hemoglobins release protons or anions upon oxygenation under the experimental conditions of the present study. The enthalpy and entropy changes are non-uniform with respect to the oxygenation step and do not need to be corrected for the oxygenation-linked release of protons and anions. For the tetrameric hemoglobin, enthalpy-entropy compensation was observed between the first, second and third steps of oxygenation. The present results suggest that the origin of the co-operative oxygenation is primarily entropic for both hemoglobin components of S. inaequivalvis. Comparison of these data with those obtained on other hemoglobins shows that no simple generalization can be made as to the thermodynamic nature of co-operativity in oxygen binding.

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

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↗