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Biomedical subjects

C Spagnuolo

Publications and source records attributed to C Spagnuolo.

At least 19 recordsLinked to original sources
PubMed2000

Illudalane sesquiterpenoids from the soft coral Alcyonium paessleri: the first natural nitrate esters.

Fifteen illudalane sesquiterpenoids, alcyopterosins A-O (1-15) have been isolated from the subAntarctic soft coral Alcyonium paessleri which was collected at a depth of 200 m near the South Georgia Islands, and their structures were elucidated by spectroscopic techniques. Eight of these compounds (2, 3, 5-8, 10, and 13) are the first natural nitrate esters, while the other four (1, 4, 11, and 12) are chlorinated. These compounds are as well the first illudalane sesquiterpenoids to be isolated from the marine environment. Compounds 1, 3, 5, and 8 showed mild cytotoxicity toward human tumor cell lines.

Animals↗
PubMed2000

Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants.

The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.

Amino Acid Substitution↗
PubMed1999

Pentacoordinate hemin derivatives in sodium dodecyl sulfate micelles: model systems for the assignment of the fifth ligand in ferric heme proteins.

Ferric iron protoporhyrin IX derivatives in SDS micelles have been investigated by means of visible absorption, resonance Raman, and XANES spectroscopies to establish specific correlations between the marker bands of the pentacoordinate derivatives obtained from the three different techniques. Hydroxyl and 1,2-dimethyl imidazole coordinated hemins display the typical spectroscopic marker bands of a pentacoordinate high-spin ferric iron derivative in both Raman and XANES spectra. In turn, the optical absorption spectra of these two derivatives are very different. This difference is in line with the assignment of hydroxyl as the fifth coordination ligand to free hemin in SDS micelles, as demonstrated by the isotopic shift of the frequency of Fe-OH bond with H(2)(18)O. The present assignments are relevant to the identification of the coordination state and the nature of the fifth ligand in ferric heme proteins.

Biophysical Phenomena↗
PubMed1996

Unusual affinity of cyanide for ferrous and ferric Scapharca inaequivalvis homodimeric hemoglobin. Equilibria and kinetics of the reaction.

The homodimeric hemoglobin from the mollusk Scapharca inaequivalvis (HbI) yields very stable ferrous and ferric cyanide adducts. The stability of the ferrous complex is particularly unusual such that it enabled determination of the spectroscopic properties of the complex and the characterization of the cyanide binding reaction to deoxygenated HbI at equilibrium and kinetically. The absorption spectrum of the ferrous cyanide complex is typical of a low-spin derivative; in the near-infrared region, it displays two bands at 695 and 840 nm attributable to charge transfer transitions. At pH 9.2, cyanide binds to deoxy HbI with no cooperativity and an apparent affinity constant of 17 M-1, which is about 10-fold higher than that for deoxy horse heart myoglobin. The rate of cyanide dissociation from both the ferrous and the ferric HbI adducts is slow relative to those of the other hemoproteins investigated to date and provides the major contribution to the unusual affinity for the ligand. The rate of cyanide binding to the ferric protein, in which the pentacoordinate derivative is the dominant species, is about 100-fold faster relative to that of the ferrous protein. In structural terms, the high affinity for cyanide of Scapharca hemoglobin has been ascribed to the decreased overall polarity of the heme pocket which is related to the localization of the heme groups at the subunit interface.

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↗
PubMed1991

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis. Reaction of the oxidized derivatives with azide and fluoride.

The reaction of the oxidized derivatives of the dimeric (HbI) and tetrameric (HbII) Scapharca inaequivalvis hemoglobins with azide and fluoride has been studied. The two oxidized hemoglobins have specific characteristics. Oxidized HbI consists of a dimeric high-spin aquomet form which is in a pH-dependent association-dissociation equilibrium with a monomeric low-spin hemichrome. In contrast, in HbII the high-spin aquomet derivative is only a transient species that converts itself into a tetrameric hemichrome which in turn dissociates into lower molecular weight forms. The reaction of oxidized HbI with azide and fluoride can be described in terms of a simple reaction scheme which assumes that external ligands bind only to the aquomet derivative. In the case of HbII, the reaction route is the same; however, the situation is complicated by the fact that in the dissociated hemichromes the internal protein ligand can no longer be displaced. Therefore, irreversible processes take place whose relevance depends primarily on the affinity of the external ligand for the ferric heme iron.

Animals↗
PubMed1988

Oxidation reaction of Scapharca inaequivalvis hemoglobins with nitrite.

The oxidation reaction with nitrite of the dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis has been studied kinetically and at equilibrium. In line with previous findings obtained with ferricyanide as oxidant, in both proteins the stable oxidation product is a hemichrome, although the nitrite-methemoglobin complex is formed in significant amount when excess nitrite is employed. The reaction kinetics are characterized by a lag period followed by an autocatalytic phase, as in the case of human hemoglobin. However, with respect to human hemoglobin, in the two molluscan proteins the lag phase is prolonged significantly due to the instability of their met-form, an obligatory intermediate for the onset of autocatalysis. All the data obtained in spectrophotometric, EPR and sedimentation velocity experiments under a variety of experimental conditions conform to the reaction mechanism proposed for human hemoglobin (Spagnuolo et al., Biochim. Biophys. Acta 911 (1987) 59-63) provided hemichrome formation and nitrite binding are taken into account.

Animals↗
PubMed1987

Oxidation reaction of human oxyhemoglobin with nitrite: a reexamination.

The oxidation reaction of human oxyhemoglobin with nitrite is complex and is characterized by a lag period followed by an autocatalytic phase. On the basis of contradictory experimental results, in order to describe the time-course of the reaction, two different mechanisms have been proposed, involving either hydrogen peroxide or the superoxide anion as reaction intermediates. This paper reports a careful reinvestigation of this reaction carried out as a function of reagent concentration, buffer composition, presence of enzymatic scavengers of oxygen radicals or of other compounds which may affect the intermediate steps of the reaction. The results obtained show that: hydrogen peroxide can be definitely identified as the reaction intermediate, in agreement with the mechanism proposed by Kosaka et al. (Biochim. Biophys. Acta 702 (1982) 237-241); the reaction time-course depends in a different way on the concentrations of hemoglobin and nitrite, a finding that cannot be explained on the basis of this mechanism. A more complex reaction scheme is proposed, that provides a satisfactory description in quantitative terms for all the available experimental data.

Buffers↗
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↗
PubMed1979

Differential effects of probenecid on the levels of endogenous PGF2 alpha and TXB2 in brain cortex.

Probenecid in single or repeated doses does not modify levels of PGF2 alpha and TXB2 in rat brain cortex. After administration of subconvulsant dose of pentamethylene tetrazole (PMT) PGF2 alpha increases sharply and rapidly declines subsequently, whereas the elevation of TXB2 is smaller but of longer duration. After probenecid pretreatment PGF2 alpha levels do not decline up to 30 minutes after the initial peak and are still elevated after 60 minutes. Levels of TXB2 tend to be reduced after pretreatment. Differences in transport process or in biosynthetic compartments for these arachidonic acid (AA) metabolites may account for the observed data.

Animals↗
PubMed1979

PGF2 alpha, thromboxane B2 and HETE levels in gerbil brain cortex after ligation of common carotid arteries and decapitation.

The effects of ligation of both common carotid arteries in the gerbil on the levels of PGF2 alpha, TXB2, HETE and of energy metabolites in brain cortex, have been investigated. Also, in the same experimental conditions the changes of cyclic AMP in brain cortex, cerebellum, striatum and hippocampus have been monitored. ATP, glycogen, glucose and phosphocreatine decrease whereas, lactate and cyclic AMP are enhanced in the ischemic brain, as previously reported. In contrast, levels of arachidonic acid metabolites are not modified. During ischemia following decapitation, instead, PGF2 alpha, and TXB2, show considerable increase.

Animals↗