Franca Ascoli

Amino acid sequence of the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis and topology of the intersubunit contacts

The dimeric hemoglobin (HbI) from Scapharca inaequivalvis is highly homologous to the other known dimeric Arcid hemoglobins. The sequence has a distinctive hydrophobicity profile in the region corresponding to the E and F helices with respect to both the hemoglobin and myoglobin chains from vertebrates due to the presence of several additional hydrophobic residues. The characteristic topology of the E and F helices is conserved in all the known sequences of Arcid hemoglobins including that of the so‐called α chain of the tetrameric component from Anadara trapezia. The rationale for this conservation lies in the unusual assembly of Arcid hemoglobins where the E and F helices are involved in the interdimeric contact. It is suggested that the extra hydrophobic residues play a major role in the assembly of the basic dimeric unit in these hemoglobins.

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.

Ligand-dependent behavior of the hemoglobin from the ascarid Parascaris equorum

Abstract Hemoglobin from the ascarid Parascaris equorum is made of eight identical subunits, each containing protoheme as a prosthetic group. The thermodynamic and kinetic properties of the binding of this protein with three ligands, i.e., oxygen, carbon monoxide and ethyl isocyanide, have been investigated. The reaction with ethyl isocyanide is characterized by a hyperbolic binding curve ( n = 1), whereas binding of CO displays a weak but definite cooperativity ( n = 1.3). Displacement experiments suggest that the noncooperative binding of ethyl isocyanide occurs fully in the high-affinity state, related to a very early quaternary switch. All three ligands display binding kinetics similar to those of T-state HbA. Dissociation rate constants for CO and ethyl isocyanide fall in the same range observed for other hemoglobins. On the other hand, oxygen affinity is much higher than in human HbA and, similarly to Ascaris lumbricoides Hb, it has to be attributed to an unusually slow dissociation rate constant. In this paper it is shown that such a slow kinetic process is not affected by: (i) pH; (ii) guanidine-HCl; (iii) a large excess of p -chloromercuribenzoate; (iv) the lifetime of the oxy-Hb complex.

Immunological properties of the dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis

The dimeric and tetrameric hemoglobins from the bivalve mollusc Scapharca inaequivalvis produce highly specific antibodies in rabbits. No antigenic cross‐reactivity between the two hemoglobins has been observed despite the presence of highly conserved regions, i.e., the E and F helices, in the dimer and in the α‐chain of the tetramer. These results may be explained on the basis of the unusual assembly of S. inaequivalvis hemoglobins, in which the E and F helices are not exposed to solvent as in vertebrate hemoglobins, but form a crucial intersubunit contact. The specific antibodies only slightly affect the oxygen binding properties of the respective antigen.

Redox potentials of normal and SH(β93)-modified human hemoglobin: Effect of pH and d-glycerate 2,3-bisphosphate

The effect of pH and D-glycerate 2,3-bisphosphate on the oxidation-reduction equilibria of human hemoglobin, normal and modified at the SH(beta 93) groups by reaction with cystine dimethylester has been studied with the aim of comparing the redox equilibria with the oxygen equilibria previously studied under the same experimental conditions. Analysis of data has shown that: (i) the same groups are involved in the oxygenation and oxidation alkaline Bohr effect; (ii) the pK values of these groups are lower in met-hemoglobin than in oxyhemoglobin; (iii) the difference in the affinity for D-glycerate 2,3-bisphosphate of ferric and deoxyhemoglobin is pH-independent in the normal protein, while significantly decreasing with a decrease in pH in the modified protein; (iv) the high cooperativity of the redox process (also occurring at acid pH) and the reduced oxidation Bohr effect observed in the modified protein are consistent with the destabilization of the T structure induced by the bulky reagent bound to the SH(beta 93) groups of the protein.

899 — A potentiometric study on the redox properties of hemoglobin from Camelus dromedarius

Abstract A potentiometric study of the redox properties of hemoglobin from Camelus dromedarius has been performed, with the aim of having a better insight in the structure-function relationships of this protein; data on human hemoglobin have been included for comparative purposes. Analysis of the experimental data has shown: 1. the existence of intermediate conformers (neither R nor T ) in the oxidized form of dromedary hemoglobin; 2. the amino acid groups involved in the oxidation Bohr effect of dromedary and human hemoglobins are likely to be the same; thus, only invariant amino acid residues seem to be involved in this effect.

Invertebrate Hemoglobins: The Dimeric Hemoglobin from the Mollusc Scapharca Inaequivalvis

The dimeric hemoglobin from the mollusc Scapharca inaequivalvis represents a beautifully simple model system for the study of cooperative phenomena in hemoglobins. The mollusc was brought to our attention due to an ecological problem. S. inaequivalvis is an indopacific species which has settled along the Middle Adriatic coast only in recent years. Its demographic increase has since been explosive and has been associated with the progressive disappearance of the native species Venus gallina. The adaptation and the survival of S. inaequivalvis can be easily explained in terms of the presence of erythrocytes in its coelomic fluid and of the periodical limitations in oxygen content of the Middle Adriatic Sea due to the process of eutrophization.1

An EPR Study of NO Bonding to Erythrocruorin from the Earthworm Octolasium Complanatum

Abstract The EPR properties of the nitric oxide derivative of Octolasium complanatum erythrocruorin have been investigated as a function of the concentration of protons and cations which are known to affect the oxygen-linked allosteric equilibrium. The EPR spectrum has a rhombic shape with gx = 2.08, gz = 2.005, and gy = 1.99, and remains unchanged under all the experimental conditions used. A supernyperfine pattern consisting of nine equally spaced lines is present in the gz region indicating an interaction with two nonequivalent nitrogen atoms, one contributed by the nitric oxide and the other by the proximal histidine. The constancy of the EPR spectrum suggests that changes in the allosteric equilibrium do not involve differences in the strain of the Fe(II)-histidine bond as in tetrameric hemoglobins.

Determination of methionine sulfoxide in proteins: comparison of a gas-chromatographic and electrophoretic method.

Two methods for the determination of methionine in proteins have been used to estimate the extent of methionine sulfoxide obtained upon exposure of proteins to oxidizing agents. Both methods are based on prior treatment with cyanogen bromide, which attacks methionines (but not the sulfoxide derivative) with the resultant formation of methyl thiocyanate and peptides. The amount of methyl thiocyanate is determined quantitatively by gas chromatography, while the number of peptides is ascertained by SDS-polyacrylamide gel electrophoresis. The gas chromatographic estimate of CH3SCN offers an accurate and precise method (down to nanogram values) for the quantitative determination of methionine sulfoxide in proteins. Due to its simplicity and the use of low-cost equipment, the electrophoretic method appears to be a valuable complement to the gas chromatographic method, and the two methods in conjunction provide novel results.

Thermodynamic characterization of the allosteric transition in trout hemoglobin

The pH induced spectral changes in the Soret region occurring in the carbomonoxy derivative of trout HbIV have been measured under carefully controlled conditions of temperature and organic phosphate concentration. Parallel experiments on the kinetics of carbon monoxide dissociation by NO replacement have been performed over the same pH range. Both sets of results agree satisfactorily with a thermodynamic scheme independently drawn on the basis of functional data previously analyzed within the framework of a two state allosteric model. Thus, the whole body of data strongly supports the idea that the spectral changes are themselves an indication of the pH induced structural transition, thereby reflecting the stabilization of the liganded T-state of the molecule at low pH. This allows us to definitely conclude that the functional changes induced in trout HbIV-CO by protons are associated with a red shift of the Soret absorption band.