Biomedical subjects

A Boffi

Publications and source records attributed to A Boffi.

At least 37 records · Page 2Linked to original sources

PubMed1998

Immobilized apo-myoglobin, a new stable reagent for measuring rates of heme dissociation from hemoglobin.

Apo-myoglobin covalently linked on CNBr-activated Sepharose 4B is proposed as a new heme acceptor for investigating the heme transfer reaction from hemoproteins. Immobilized apo-myoglobin has the desirable properties of an ideal heme acceptor in that it is characterized by a high affinity for ferric heme, a high stability towards denaturation even at physiological temperatures and can be lyophilized for long-term storage. The study of heme release from myoglobin at pH 5.0 and 37 degrees C indicates that heme affinity is increased at least 10-fold relative to the soluble protein. Experiments with human hemoglobin allowed the estimation of the heme release rates from both alpha and beta chains and brought out the greater temperature sensitivity of the alpha chain heme-globin linkage.

Apoproteins↗

PubMed1997

Near infrared absorption spectra of human deoxy- and oxyhaemoglobin in the temperature range 20-40 degrees C.

Temperature-dependent spectral changes of human haemoglobin A (HbA) derivatives were investigated in the range 20-40 degrees C. The intensity of the deoxy-HbA decreased by 3-3.5%, while that of oxy- and met-HbA by less than 1%, when the temperature increased from 20 degrees to 40 degrees C. The present findings can be employed to improve the algorithms used in in vivo near infrared spectroscopy.

Hemoglobins↗

PubMed1997

Circular dichroism spectroscopy of Lucina I hemoglobin.

The monomeric hemoglobin from the mollusc Lucina pectinata (HbI) represents an interesting model system for the study of heme-related circular dichroic (CD) bands in view of the highly asymmetric distribution of aromatic residues around the heme pocket revealed by the X-ray crystal structure. The CD spectra of both ferrous and ferric HbI derivatives exhibit negative CD bands in the Soret and ultraviolet region with an enhanced ellipticity of the heme N and L bands in the near-UV region. In contrast, the magnitude of the Cotton effect in the visible and Soret regions is comparable to that observed in other hemoproteins. The spectrum of the carbon monoxide derivative shows a surprising similarity with that observed for the soybean leghemoglobin carbon monoxide adduct. A common structural feature in the two proteins is the presence in the distal pocket of two Phe residues (B9 and B10) the aromatic rings of which are perpendicular to the heme plane.

Animals↗

PubMed1997

Stereochemistry of the Fe(II)- and Fe(III)-cyanide complexes of the homodimeric Scapharca inaequivalvis hemoglobin. A resonance Raman and FTIR study.

Resonance Raman measurements carried out in parallel on ferrous and ferric Scapharca inaequivalvis dimeric hemoglobin cyanide derivatives allowed the identification of the electron density marker bands and heme core size marker bands in both derivatives in comparison with those obtained for the carbonmonoxy and deoxy adducts. The iron cyanide stretching mode, measured for the first time in a ferrous hemoprotein, has been detected at 455 cm(-1), only 6 cm(-1) lower than in the corresponding ferric derivative. This finding demonstrates that the large free energy difference for complex formation between the two derivatives is not concentrated on the Fe-C bond. The internal stretching frequencies of the ligand in the ferric and ferrous derivatives have been identified by FTIR and Raman measurements using different cyanide isotopes. The frequency decreases in the order Fe(III)-CN adduct, free cyanide, Fe(II)-CN adduct, consistent with the behavior observed in inorganic complexes and horseradish peroxidase. The main feature emerging from these data is that cyanide, at variance with oxygen and carbon monoxide, binds to ferrous iron with only a minor perturbation of the electronic structure of the heme. The functional counterpart of this effect is the absence of cooperative cyanide binding in HbI.

Animals↗

PubMed1997

Dynamics of cyanide binding to ferrous Scapharca inaequivalvis homodimeric hemoglobin.

Flash photolysis experiments have been carried out for the first time on a hemoglobin ferrous cyanide adduct with an 8 ns laser pulse. A 95% nonexponential rebinding process occurs within 2 micros after full photolysis in ferrous cyanide dimeric Scapharca inaequivalvis hemoglobin (HbI), indicating that once photolyzed the cyanide anion is not able to escape from the protein matrix and rebinds to the heme iron. The resonance Raman spectrum of the 10 ns photoproduct is identical to that of the fully relaxed deoxy derivative, indicating that in the ferrous cyanide HbI adduct protein relaxation occurs within 10 ns after photolysis. This behavior is at variance with that of the carbonmonoxy HbI derivative in which very little geminate rebinding is observed and the photoproduct relaxes with a lifetime of 1 micros. The fast relaxation of the cyanide HbI photoproduct can be accounted for by the small perturbation of the heme structure induced by cyanide binding to ferrous HbI. This is consistent with a deoxy-like conformation of the HbI ferrous cyanide adduct and implies that the pathway for relaxation involves only minor local rearrangements of the heme moiety. Photolysis experiments carried out on ferrous cyanide horse myoglobin, which can be saturated only partially, show a qualitatively similar behavior in ligand rebinding, indicating that the geminate process of the cyanide anion is a general phenomenon in hemoproteins.

Animals↗

PubMed1997

Effect of the vinyl-globin interactions on the temperature-dependent broadening of the Soret spectra: a study with horse myoglobin and Scapharca dimeric hemoglobin reconstituted with unnatural 2,4-heme derivatives.

The temperature dependence of the Soret absorption spectra has been measured over the range 80 to 300 K on deoxygenated and carbonmonoxy horse heart myoglobin and Scapharca inaequivalvis dimeric hemoglobin reconstituted with proto- or with meso- and deutero-heme, in which the vinyl groups have been replaced with ethyl groups or hydrogen atoms, respectively. In the meso- and deutero-derivatives of both proteins the linewidth of the absorption spectra is narrower and less sensitive to thermal broadening effects than in the proto-derivatives. Moreover, the broadening effects are larger in the deoxygenated proteins with respect to the liganded adducts. The quantitative analysis of these effects shows that the change in linewidth is due to a marked decrease in the extent of coupling between the heme vibronic transitions and the protein low-frequency motions. The relevance of the vinyl groups in the dynamics of the heme-globin interaction is highlighted by this experimental approach which shows that the protein is capable of transmitting structural information to the heme by coupling the ensemble of the low-frequency modes to the stereochemistry of the vinyl itself. This mechanism, which entails adjustment of the equilibrium between vinyl torsional conformers, represents an additional pathway for the control of the heme reactivity in addition to the iron-histidine link.

Animals↗

PubMed1997

The unique heme-heme interactions of the homodimeric Scapharca inaequivalvis hemoglobin as probed in the protein reconstituted with unnatural 2,4 heme derivatives.

In the homodimeric hemoglobin from Scapharca, HbI, functional communication between the two heme groups is based on their direct structural linkage across the subunit interface through the heme propionates. The heme-protein interactions have been altered in deutero- and meso-HbI by substituting the vinyl groups at positions 2 and 4 of protoheme with hydrogen and ethyl groups, respectively. In meso-HbI the introduction of the ethyl groups in the heme pocket induces significant alterations in the conformation of the heme peripheral substituents, including the propionates, and in the structure of bound CO, as revealed by the resonance Raman spectra. The functional counterpart of these structural changes is the loss of cooperativity in carbon monoxide binding and in the rate of oxygen dissociation. Oxygen pulse and flash photolysis experiments indicate that meso-HbI is locked in the liganded conformation. It is postulated that the ethyl groups, which occupy a larger volume than vinyl ones, impair the ligand-linked movement of the heme relative to its pocket and in turn the expression of cooperativity. In deutero-HbI structural alterations have not been monitored. Functionally, cooperativity in the CO binding kinetics is increased as if hydrogen atoms at positions 2 and 4 permitted more marked movements of the heme than in the native protein.

Animals↗

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↗

PubMed1996

Stability of the heme-globin linkage in alphabeta dimers and isolated chains of human hemoglobin. A study of the heme transfer reaction from the immobilized proteins to albumin.

The stability of the heme-globin linkage in alphabeta dimers and in the isolated chains of human hemoglobin has been probed by studying the transfer of heme from the proteins immobilized onto CNBr-activated Sepharose 4B to human albumin. The kinetic and equilibrium features of the reaction have been measured spectrophotometrically given the stability of the heme donors and the ease with which heme donor and acceptor can be separated. Isolated alpha and beta chains transfer heme to albumin at similar rates (1 6 x 10(-2) s-1 at pH 9.0 and 20 degrees C) in the ferrous CO-bound and in the ferric state. In alpha beta dimers the heme-globin linkage is strengthened considerably, albeit to a different extent in the ferrous CO-bound and ferric met-aquo derivatives. Only in the latter heme is lost at a measurable rate, 0.065 +/- 0.011 x 10(-2) s-1 for alpha heme and 2.8 +/- 0.6 x 10(-2) s-1 for beta heme at pH 9.0 and 20 degrees C, which is very close to the rate measured with soluble met-aquo-hemoglobin at micromolar concentrations. These results indicate that in human hemoglobin the heme-globin linkage in the alpha chains is stabilized by interactions between unlike chains at the alpha1 beta1 interface, whereas heme binding to the beta chains is stabilized by interactions at the alpha1beta2 interface. These long range factors have to be taken into account in addition to the local factors at the heme pocket when evaluating the effect of point mutation and chemical modification.

Albumins↗

PubMed1996

Near-infrared spectra of Scapharca homodimeric hemoglobin: characterization of the deoxy and photodissociated derivatives.

The near-infrared charge transfer band at 760 nm (band III) has been investigated in deoxy and photodissociated dimeric Scapharca hemoglobin. At 300 K, the 10-ns spectrum of the carbonmonoxy derivative photoproduct is shifted by about 6 nm toward longer wavelengths with respect to the deoxy spectrum, both in buffer and in glycerol/buffer solutions. Moreover, the band III peak occurs at about the same wavelength at 300 K and at 10 K for the 10-ns photodissociated derivative, whereas in the deoxy derivative large changes in peak position and linewidth are observed as a function of temperature. These findings suggest that in dimeric Scapharca hemoglobin the photoproduct has not relaxed after 10 ns. The complete time dependence of the relaxation process has been studied both in buffer and in glycerol/buffer solutions at room temperature. The relaxation from the photoproduct to the deoxy species occurs on a microsecond time scale, in line with recent optical absorption and resonance Raman measurements.

Animals↗

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

[Colonic ulcer in HIV (+) patients].

UNLABELLED: A characteristic rectocolonic endoscopic finding is the presence of ulcers, which may correspond to different diagnosis. Between January, 1989 and May, 1993, we performed 28 colonoscopies and 12 rectoscopies in patients with HIV (+). In all cases, histologic, parasitologic, bacteriologic, virologic, and micrologic studies were practised on tissue biopsies. Eighteen of them presented rectocolonic ulcers (we are not including anal and terminal ileum ulcers). The studies involved 16 males and 2 females, with an average age of 31, 21 years old (20-47), 14 of which were male homosexuals, 2 intravenous drug abusers, 1 hemophilic and an heterosexual female. Symptoms were as follows: 15 with hematochezia, 7 with proctorrhagia, 6 with enterorrhagia and 2 with both manifestations. All the rest, presented chronic diarrhea, loss of weight, anal secretion, asthenia, fever, constipation and anemia in isolated cases. Diagnosis were 6 rectocolonic cytomegalovirus (all occurred during the current year). Other related opportunistic pathogens included an intestinal TBC, a rectal Herpes and a Burkitt's lymphoma. Related etiological agents were not found in 9 patients, in spite of systematic quest. Opportunistics found in three of the cases revealed AIDS in those patients. COMMENTS: 1-The indexes of male homosexuals are strikingly higher than the rest of our HIV endoscopy statistics. 2-We observed prevailing idiopathic lesions. 3-The relationship between "CU like" pictures, already described, and the immunocomponents of the idiopathic CU is unknown. 4-All our CMV cases are recent. 5-Hematochezia was the most frequent symptom. 6- Herpetic ulcers correspond to HSV type 1. 7-Diffuse ulcers may correspond to lymphoma.

Adult↗

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

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

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

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↗