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E Chiancone

Publications and source records attributed to E Chiancone.

At least 55 records · Page 3Linked to original sources
PubMed1995

The 2.0 A crystal structure of Scapharca tetrameric hemoglobin: cooperative dimers within an allosteric tetramer.

The crystal structure of the allosteric tetrameric hemoglobin from Scapharca inaequivalvis (HbII) has been determined in the carbonmonoxy liganded state using a combination of anomalous scattering and molecular replacement. The molecular model has been refined at 2.0 A resolution to a conventional R-factor of 0.173 and a free R-factor of 0.244. The tetramer is formed from two identical heterodimers. Each heterodimer is assembled with intersubunit contacts involving the E and F helices and heme groups in a manner that is very similar to that of the cooperative Scapharca homodimeric hemoglobin. In addition, the ordered water structure observed in these dimeric interfaces is quite similar. These structural similarities strongly suggest that the dimers within the Scapharca tetramer are cooperative. Subunits assemble into a tetramer in a distinctly non-tetrahedral arrangement, with the pseudo 2-fold axes of the heterodimer oriented at an angle of 74.5 degrees relative to the molecular 2-fold. This arrangement requires that two subunit types have distinct locations and contacts, despite the very similar tertiary structures. HbII polymerizes to higher-order assemblages in a ligand, proton and anion dependent fashion. The lattice contacts in the HbII-CO crystal suggest possible modes for this association.

Allosteric Site↗
PubMed1995

Preparative and analytical applications of immobilized haemoglobin.

New applications of haemoglobin immobilized on Sepharose 4B are proposed for the removal of cell-free, potentially toxic haemoglobin from either natural biological specimens or artificial haemoglobin-containing systems (e.g., blood substitutes, haemosomes) to be employed for biomedical purposes. In a model study, an affinity column of immobilized haemoglobin was used to remove free haemoglobin from blood serum in which controlled haemolysis had been induced. The affinity column retains all the free haemoglobin, does not retain the haemoglobin-haptoglobin complex(es) and leaves the composition of the serum samples unaltered. When immobilized met-haemoglobin is used, haem is transferred readily to albumin, with which it forms a complex. This observation on the one hand shows that immobilized oxyhaemoglobin should be preferred for preparative purposes, and on the other opens the way to the characterization of the haem transfer reaction to albumin by means of immobilized met-haemoglobin. This reaction is difficult to study in solution owing to the overlap of the met-haemoglobin and methaemalbumin spectra.

Cell-Free System↗
PubMed1995

Rigidity of the heme pocket in the cooperative Scapharca hemoglobin homodimer and relation to the direct communication between hemes.

The Soret spectra of the dimeric hemoglobin from Scapharca and of horse myoglobin reconstituted with protoporphyrin IX and Zn-protoporphyrin IX have been measured over the range 290-80 K. With increase in temperature the Soret band broadens and shifts to a different extent depending on the protein and the presence of the metal. In the Zn-protoporphyrin IX derivatives the spectral changes are more marked in myoglobin than in the dimeric hemoglobin. In the protoporphyrin IX derivatives, in which the spectral changes are significantly reduced, the opposite is true. The data have been analyzed in terms of coupling of the protein low-frequency vibrational motions to the porphyrin electronic transition (V. Srajer et al., 1986, Phys. Rev. Lett. 57, 1267-1270; A. Di Pace et al., 1992, Biophys. J. 63, 475-484). The analysis indicates that the heme pocket of the dimeric hemoglobin is characterized by an unusual rigidity and that the metal plays a different role in the transmission of the protein motions to the heme moiety in the dimeric hemoglobin and in myoglobin. Static and dynamic fluorescence measurements carried out at room temperature are in line with these conclusions.

Allosteric Regulation↗
PubMed1995

Calcium-dependent translocation of sorcin to membranes: functional relevance in contractile tissue.

Sorcin, a 22 kDa calcium binding protein present in abundance in cardiac tissue and in multi-drug resistant cells and previously described as a soluble protein, is now shown to undergo a calcium-dependent translocation process from the cytosol to cellular membranes in both systems. The translocation process takes place also in E. coli BL21 cells that express recombinant sorcin, r-sorcin, and can be exploited in the purification of the protein. Calcium binding to purified r-sorcin occurs at micromolar concentrations of the metal and is accompanied by a conformational change that renders the protein soluble in the non-ionic detergent Triton X-114. This finding suggests that lipids are the target of sorcin on cellular membranes. The possible significance of the calcium-dependent translocation of sorcin in the specialized functions of sorcin-expressing cells is discussed.

Animals↗
PubMed1994

Structural characterization of oxidized dimeric Scapharca inaequivalvis hemoglobin by resonance Raman spectroscopy.

Resonance Raman spectra of the ferric homodimeric hemoglobin from Scapharca inaequivalvis have been measured over the pH range 5.8-8.3 in buffers of ionic strengths 0.01 and 0.1 M to determine the spin and coordination state of the iron atom. Three species contribute to the spectra: a low spin hexacoordinate, a high spin pentacoordinate, and a high spin hexacoordinate component. Optical absorption and EPR spectra measured under the same conditions allowed the identification of the ligands in the sixth coordination position, namely the distal histidine in the low spin derivative and a water molecule in the high spin one. The relative concentrations of these three species depend on pH in an unusual way. Thus, the aquomet derivative is present over the whole pH range, albeit in small amounts as most of the hemoglobin converts to the low spin hemichrome at acid pH values and to the pentacoordinate derivative at neutral and slightly alkaline ones. The formation of a pentacoordinate heme as the pH is increased has not been reported previously for other myoglobins and hemoglobins. Low ionic strength and high protein concentration favor the formation of the high spin pentacoordinate species, while at high ionic strength and low protein concentration the low spin hexacoordinate species prevails. Ionization of the iron-bound water molecule occurs at pH > or = 9.3; accordingly, signals from the hydroxyl derivative were not observed in the Raman spectra over the pH range studied.

Animals↗
PubMed1994

Coordination and spin state equilibria as a function of pH, ionic strength, and protein concentration in oxidized dimeric Scapharca inaequivalvis hemoglobin.

The oxidized homodimeric Scapharca inaequivalvis hemoglobin undergoes changes in coordination and spin state as a function of pH, ionic strength, and protein concentration which have been monitored by optical absorption spectroscopy. Three species contribute to the spectra between pH 5.8 and 8.7: (i) a hexacoordinate high spin aquomet derivative, whose concentration is essentially constant over the whole pH range analyzed; (ii) a pentacoordinate high spin component which prevails at alkaline pH values, and (iii) a hexacoordinate low spin hemichrome, which is formed at acid pH. The contribution of each of the components to the observed spectra was calculated with the singular value decomposition procedure and has been described quantitatively in terms of a linkage scheme which accounts for the change in heme coordination and for the observation that the high spin to low spin transition entails dissociation into monomers. An important feature of the linkage scheme is the cooperative binding of protons to aquomet dimers. Stopped flow experiments to study the kinetics indicate that dissociation into monomers is the rate-limiting process. The unusually strong tendency of oxidized HbI to loose the heme-bound water molecule is discussed in terms of strain in the iron-proximal histidine bond.

Animals↗
PubMed1994

Identification of the site of ferrocyanide binding involved in the intramolecular electron transfer process to oxidized heme in Scapharca dimeric hemoglobin.

The cooperative homodimeric hemoglobin (HbI)2 from the mollusc Scapharca inaequivalvis is characterized by unusual properties of the ferric derivative. The dimeric aquomet form undergoes a pH-dependent reversible dissociation into a monomeric low-spin hemichrome. Moreover, in HbI oxidized with ferricyanide the ferrocyanide anion produced in the reaction remains bound to the oxidized protein with high affinity and forms an intramolecular redox couple with the heme iron. Thus, the reduced HbI-CO adduct is obtained readily in the presence of carbon monoxide. The ferrocyanide binding site of HbI has been identified by modifying the only cysteine residue of the polypeptide chain, Cys 92 (F2), which is located at the subunit interface near the proximal histidine (His 101, F11). In HbI modified with organomercurials the rate of oxidation by ferricyanide depends on the presence and position of a negatively charged group on the aromatic ring, indicating that the binding site of the ferrocyanide anion is located near Cys 92. The tendency to dissociate into the monomeric hemichrome of the various Cys 92-reacted proteins and the study of the intramolecular electron transfer reaction between bound ferrocyanide and the heme iron confirmed this location. The proposed binding site of the ferrocyanide anion comprises a cluster of positive charges at the subunit interface formed by Lys 96, Arg 53', Lys 65', and Arg 67' where apices indicate residues of the contralateral subunit.

Animals↗
PubMed1994

The dimer-tetramer equilibrium of recombinant hemoglobins. Stabilization of the alpha 1 beta 2 interface by the mutation beta(Cys112-->Gly) at the alpha 1 beta 1 interface.

The dimer-tetramer association constants of several recombinant human hemoglobins (in the CO form) have been measured by differential gel filtration. Recombinant human hemoglobin prepared from recombinant beta-chains, and mutant hemoglobins where the substitution was on the surface, beta(Thr4-->Asp), in the heme pocket, beta(Val67-->Thr), at the 2,3-DPG binding site, beta(Val1-->Met+His2del), had a twofold smaller association with respect to natural hemoglobin. In a mutant at the alpha 1 beta 2 interface, beta(Cys93-->Ala), the association constant was decreased three-fold. Conversely, in a mutant at the alpha 1 beta 1 interface, beta(Cys112-->Gly), the association constant was two- and four-fold increased with respect to natural and recombinant human hemoglobin. These differences are energetically very small, consistent with the correct folding of the recombinant hemoglobins. The stabilization of the tetrameric structure by a mutation at the alpha 1 beta 1 interface indicates that structural changes in this interface can be propagated through the protein to the alpha 1 beta 2 interface and, thereby, exert an effect on the allosteric equilibrium.

2,3-Diphosphoglycerate↗
PubMed1994

Stereodynamic properties of the cooperative homodimeric Scapharca inaequivalvis hemoglobin studied through optical absorption spectroscopy and ligand rebinding kinetics.

The study of the thermal evolution of the Soret band in heme proteins has proved to be a useful tool to understand their stereodynamic properties; moreover, it enables one to relate protein matrix fluctuations and functional behavior when carried out in combination with kinetic experiments on carbon monoxide rebinding after flash photolysis. In this work, we report the thermal evolution of the Soret band of deoxy, carbonmonoxy, and nitric oxide derivatives of the cooperative homodimeric Scapharca inaequivalvis hemoglobin in the temperature range 10-300 K and the carbon monoxide rebinding kinetics after flash photolysis in the temperature range 60-200 K. The two sets of results indicate that Scapharca hemoglobin has a very rigid protein structure compared with other hemeproteins. This feature is brought out i) by the absence of nonharmonic contributions to the soft modes coupled to the Soret band in the liganded derivatives, and ii) by the almost "in plane" position of the iron atom in the photoproduct obtained approximately 10(-8) s after dissociating the bound carbon monoxide molecule at 15 K.

Animals↗
PubMed1993

Nucleation of the iron core occurs at the three-fold channels of horse spleen apoferritin: an EXAFS study on the native and chemically-modified protein.

Extended X-ray absorbance fine structure measurements have been carried out on the initial Fe(III)-apoferritin complex at a Fe/subunit ratio of 2 in native and modified horse spleen apoferritin. Analysis of the data indicates that in the native protein the iron forms a protein-bound polynuclear cluster (Fe-Fe distance 3.4 A) with a first coordination sphere constituted by 5-6 low-Z atoms, e.g., nitrogen atoms, carboxylate-like ligands or oxo bridges between the iron atoms. Modification of Cys-126, a residue localized on the outer surface of the hydrophilic three-fold channels, with p-chloromercuribenzoate (PMB) or phenylmercuric acetate (PMA) brings about distinctive differences. In particular, in the PMB-reacted protein the feature assigned to the iron-iron interaction disappears from the spectrum, whilst in the PMA-reacted protein the main differences with respect to the native protein are observed at the level of the first coordination sphere. These results confirm the formation of protein-Fe(III)-clusters and localize these sites at the hydrophilic three-fold channels of horse spleen apoferritin.

Animals↗
PubMed1993

Protein-heme interactions in hemoglobin from the mollusc Scapharca inaequivalvis: evidence from resonance Raman scattering.

Resonance Raman spectra of the Scapharca inaequivalvis homodimeric hemoglobin (HbI) have been measured for the ligand-bound and ligand-free ferrous forms of the protein. In the deoxy derivative, the iron-histidine (Fe-His) stretching mode, proposed as a marker of the oxygen affinity and a conduit linking the hemes to the subunit interface, gives rise to a Raman peak centered at 203 cm-1, an unusually low frequency compared to that reported for other hemoglobins and myoglobins. In the CO-bound derivative, three isotope-sensitive lines at 517, 583, and 1945 cm-1 have been assigned to the Fe-CO stretching, Fe-C-O bending, and C-O stretching modes, respectively. From the frequencies of these modes and from their relative intensities, the Fe-C-O geometry appears to be tilted from axial coordination and shows a bending angle which has been estimated to be about 171 +/- 5 degrees. For the oxygen derivative, only one isotope-sensitive peak has been detected at 570 cm-1, in line with the values found for myoglobin and other hemoglobins. Resonance Raman spectra of HbI modified with p-(chloromercuri)benzoate (PMB) at Cys92 have been measured in parallel with those of the native protein. Despite the large increase in oxygen affinity produced by the PMB modification, the frequency of the Fe-His stretching mode is unshifted in the deoxy derivative.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Ligand binding and conformation change in the dimeric hemoglobin of the clam Scapharca inaequivalvis.

The reaction with carbon monoxide of the cooperative dimeric hemoglobin from Scapharca inaequivalvis has been examined by flash photolysis. In the nanosecond time range, geminate rebinding of 5% of dissociated CO occurs with a rate constant of 1.4 x 10(7) s-1. There is a change in absorbance of deoxyhemoglobin following photolysis at a rate of 1.2 x 10(6) s-1, consistent with a shift in the position of the Soret band to longer wavelengths. The amplitude of the change is proportional to the population of deoxydimer. In much of the Soret region this change is greater than the absorbance excursion associated with geminate recombination. There is at least one other slower change associated with the singly liganded species. Geminate rebinding of NO has components of 50, 8, and 0.035 ns-1, accounting for 75%, 25%, and less than 1% of the total reaction observed after a 35-ps photolysis flash. Simulation of diffusion of NO by molecular dynamics shows the ligands moving from the heme pocket to a subsidiary space between the edge of the heme and the surface of the protein.

Animals↗
PubMed1993

Heme-heme interactions in a homodimeric cooperative hemoglobin. Evidence from transient Raman scattering.

The comparison of the resonance Raman spectrum of the deoxy dimeric hemoglobin (HbI) from the Arcid clam, Scapharca inaequivalvis, to its CO photoproduct at 10 ns reveals several significant differences in the low frequency vibrational modes including those involving motions of the peripheral substituents on the heme such as the propionates. This finding reflects the involvement of the propionates in a hydrogen bonding network which connects the two heme groups across the subunit interface and is sensitive to ligand binding. A frequency difference in the iron-histidine stretching mode of 6 cm-1 in this invertebrate hemoglobin is substantially smaller than that detected in tetrameric vertebrate hemoglobins under similar conditions. Time evolution studies of the vibrational modes from the photoproduct demonstrate that the transient form relaxes to the deoxy structure concertedly with a half-life of 1 microseconds, the time scale in which tertiary relaxations of tetrameric hemoglobins occur. These data establish that the tertiary and quaternary structural changes take place on the same time scale and confirm a mechanism of cooperativity involving direct interaction between the two heme groups through the peripheral substituents. This direct structural communication provides a very tight linkage between the heme groups prohibiting modulation of the oxygen affinity by factors in the physiological milieu.

Animals↗
PubMed1993

Metastable CO binding sites in the photoproduct of a novel cooperative dimeric hemoglobin.

The infrared absorption spectrum of the CO-photoproduct from Scapharca inaequivalvis hemoglobin (Hbl) at 10 K yields only a single line in the "B" state region at 2132 cm-1. This is the same frequency as the B1 line observed in photodissociated vertebrate HbCO and MbCO. No evidence was found for the B2 line detected in vertebrate hemoglobins and myoglobin in the 2118-2120 cm-1 region. These data demonstrate that the protein does not have the same conformationally accessible ligand-binding sites as do vertebrate hemoglobins and myoglobins. The absence of the B2 line indicates that only a single weak site is accessible to the photolyzed CO molecule. These results are in accord with geminate rebinding experiments and ligand escape pathway calculations which have shown that the distal properties of Hbl are distinct from those of tetrameric hemoglobins and vertebrate myoglobins.

Animals↗
PubMed1993

Selective removal of beta-lactoglobulin directly from cow's milk and preparation of hypoallergenic formulas: a bioaffinity method.

beta-Lactoglobulin, a major allergen of cow's milk, can pass into the breast milk of mothers consuming dairy products and thus sensitize a predisposed infant with a family history of atopic allergy. beta-Lactoglobulin coupled to Sepharose 4B can be used as a specific affinity matrix to remove beta-lactoglobulin from milk without the use of buffer systems. The tendency of beta-lactoglobulin to polymerize reversibly in milk promotes binding of the soluble protein to the coupled protein and a significant retardation in its elution with respect to the other milk proteins. The matrix can be regenerated with water and used repeatedly. These unique characteristics can be exploited in bioreactors designed to treat milk for the preparation of new hypoallergenic hyposensitizing milk formulas to be used by lactating mothers.

Allergens↗
PubMed1992

The homodimeric hemoglobin from Scapharca can be locked into new cooperative structures upon reaction of Cys92, located at the subunit interface, with organomercurials.

In the cooperative, homodimeric hemoglobin from Scapharca inaequivalvis, HbI, the subunit interface is formed by the heme-carrying E and F helices and contains the only cysteine residue of the globin chain (Cys92, F2) in an area which changes from hydrophilic to hydrophobic upon oxygenation. Binding of organomercurials to HbI is cooperative and entails major quaternary rearrangements. The reaction of Cys92 with p-chloromercuri-benzoate (PMB) and p-nitro-o-chloromercuriphenol (PN), a sensitive reporter of the cysteine microenvironment at neutral pH values, has been followed in stopped flow experiments. Kinetic evidence for the cooperativity of mercurial binding has been obtained and the rate of the corresponding conformational transition has been estimated. As expected PN, but not PMB, is able to monitor the oxygen-linked change of the cysteine microenvironment. The modification of Cys92 with PN has unique functional effects. In PN-reacted HbI cooperativity is maintained, albeit to a different extent, depending on the ligation state of the protein during mercaptide formation. It may be envisaged that PN locks the protein into new, cooperative, quaternary structures stabilized by hydrogen bonding interactions between the ionized nitrophenol moiety and the contralateral subunit.

Animals↗
PubMed1992

Immobilized hemoglobin in the purification of hemoglobin-based oxygen carriers.

Chemically modified hemoglobins can be used as oxygen carriers in cell-free fluids provided that they have a low oxygen affinity and are stable towards dissociation into subunits. The latter species are undesirable because they are filtered rapidly through the kidneys, have renal toxicity and are characterized by a high oxygen affinity. A most important step in the preparation of hemoglobin-based oxygen carriers is therefore their purification from any dissociable material. Hemoglobin immobilized as alpha beta dimers on Sepharose lends itself naturally to this purpose as it is able to interact in a specific and reversible way with soluble alpha beta dimers. Hemoglobin affinity columns are very effective in the purification of cross-linked and pseudo-cross-linked human and bovine hemoglobin. The applicability of the technique is enhanced by the ease with which alpha beta dimers from different species cross-interact to yield hybrid alpha 2 beta 2 tetramers. It is shown that hemoglobin affinity columns may provide analytical information on the cross-linking reaction itself.

Animals↗