Search PubMed⌕ Search

Biomedical subjects

A Cupane

Publications and source records attributed to A Cupane.

At least 37 records · Page 2Linked to original sources
PubMed1994

Thermal broadening of the Soret band in heme complexes and in heme-proteins: role of iron dynamics.

We report the thermal broadening of the Soret band in heme-CO, heme-OH and protoporphyrin IX in the temperature range 300-20 K. For protoporphyrin IX the temperature dependent Gaussian line broadening follows the behavior predicted by the harmonic approximation in the entire temperature range investigated. In contrast, for heme-CO and heme-OH the harmonic behavior is obeyed only up to about 180 K and an anomalous line broadening increase is observed at higher temperatures. This effect is attributed to the onset of anharmonic motions of the iron atom with respect to the porphyrin plane. Comparison with previously reported analogous data for heme proteins enables us to suggest that the onset of substrate interconversions in these latter systems can be reflected in motions of the iron atom with respect to the porphyrin plane.

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 dynamics: conformational disorder, vibrational coupling and anharmonicity in deoxy-hemoglobin and myoglobin.

In this work we study the temperature dependence of the Soret band lineshape of deoxymyoglobin and deoxyhemoglobin, in the range 300-20 K. To fit the measured spectra we use an approach originally proposed by Champion and coworkers (Srajer et al. 1986; Srajer and Champion 1991). The band profile is modelled as a Voigt function that accounts for the coupling with low frequency vibrational modes, whereas the coupling with high frequency modes is responsible for the vibronic structure of the spectra. Moreover, owing to the position of the iron atom out of the mean heme plane, inhomogeneous broadening brings about a non-Gaussian distribution of 0-0 electronic transition frequencies. The reported analysis enables us to isolate the various contributions to the overall bandwidth, and their temperature dependence points out the relevance of low frequency vibrations and of large scale anharmonic motions starting at temperatures higher than 170 K. Information on the mean iron-heme plane distance and on its temperature dependence, as well as on the heme pocket conformational disorder, is also obtained.

Animals↗
PubMed1993

Structure-dynamics-function relationships in Asian elephant (Elephas maximus) myoglobin. An optical spectroscopy and flash photolysis study on functionally important motions.

In this work we report the thermal behavior (10-300 K) of the Soret band lineshape of deoxy and carbonmonoxy derivatives of Asian elephant (Elephas maximus) and horse myoglobins together with their carbon monoxide recombination kinetics after flash photolysis; the results are compared to analogous data relative to sperm whale myoglobin. The Soret band profile is modeled as a Voigt function that accounts for the coupling with high and low frequency vibrational modes, while inhomogeneous broadening is taken into account with suitable distributions of purely electronic transition frequencies. This analysis makes it possible to isolate the various contributions to the overall lineshape that; in turn, give information on structural and dynamic properties of the systems studied. The optical spectroscopy data point out sizable differences between elephant myoglobin on one hand and horse and sperm whale myoglobins on the other. These differences, more pronounced in deoxy derivatives, involve both the structure and dynamics of the heme pocket; in particular, elephant myoglobin appears to be characterized by larger anharmonic contributions to soft modes than the other two proteins. Flash photolysis data are analyzed as sums of kinetic processes with temperature-dependent fractional amplitudes, characterized by discrete pre-exponentials and either discrete or distributed activation enthalpies. In the whole temperature range investigated the behavior of elephant myoglobin appears to be more complex than that of horse and sperm whale myoglobins, which is in agreement with the increased anharmonic contributions to soft modes found in the former protein. Thus, to satisfactorily fit the time courses for CO recombination to elephant myoglobin five distinct processes are needed, only one of which is populated over the whole temperature range investigated. The remarkable convergence and complementarity between optical spectroscopy and flash photolysis data confirms the utility of combining these two experimental techniques in order to gain new and deeper insights into the functional relevance of protein fluctuations.

Animals↗
PubMed1992

Strong vibronic coupling in heme proteins.

We report the near infrared absorption spectra of cyanomethemoglobin and cyanometmyoglobin in two different solvents (deuterated solutions containing 65% v/v glycerol(OD)3 or 65% v/v ethylene glycol(OD)2). At 25 K the spectra show a clearly resolved fine structure that can be accounted for by considering a strong coupling of the porphyrin-to-iron charge transfer transitions with a single vibrational mode at 365 cm-1. The coupling constants depend on both the specific electronic transition and the protein surrounding the chromophore, indicating once more the specificity of heme globin interactions.

Humans↗
PubMed1992

Protein dynamics. Vibrational coupling, spectral broadening mechanisms, and anharmonicity effects in carbonmonoxy heme proteins studied by the temperature dependence of the Soret band lineshape.

In this work we study the temperature dependence of the Soret band lineshape of the carbonmonoxy derivatives of sperm whale myoglobin, human hemoglobin, and its isolated alpha and beta subunits. To fit the observed spectral profile we use an analytic expression derived for a system whereby a single electronic transition is coupled to Franck-Condon active vibrational modes, within the adiabatic and harmonic approximation. The vibronic structure of the spectra arises from the coupling with high frequency modes; these modes contribute to the total line shape through a series of Lorentzians with peak positions at vibrational overtones and half width related to the time constant of the population decay of the excited electronic state (homogeneous broadening); moreover, the coupling with low frequency modes broadens each Lorentzian to a Voigtian. Inhomogeneous broadening is modeled as a gaussian distribution of the 0-0 transition frequencies and is therefore added as a constant term to the previous gaussian width. This spectral deconvolution enables us to investigate the different contributions to line broadening and the parameters that characterize the vibrational coupling, as well as their dependence upon protein and solvent composition. The investigation is carried out as a function of temperature in the range 20-300 K; relevant information is obtained by comparing experimental results with theoretical predictions. This work supports a description of the investigated proteins as heterogeneous systems, whose heterogeneity depends on the particular protein and on the composition of the external matrix. The delocalized pi electron cloud of the porphyrin ring is coupled not only to the high frequency vibrational modes of the active site but also to a "bath" of lower frequency modes that involve the entire protein; moreover at suitable temperatures (approximately 200 K), anharmonic motions, which are an obvious prerequisite for the jumping among different conformational substates, become evident.

Animals↗
PubMed1991

The effect of organic cosolvents on the oxygen affinity of fetal hemoglobin. Relevance of protein-solvent interactions to the functional properties.

We have studied the effects of organic cosolvents (monohydric alcohols and formamide) on the oxygen affinity of human fetal hemoglobin stripped of phosphates and have compared them with the effects of the same cosolvents on the oxygen affinity of human adult hemoglobin under the same experimental conditions. Our results confirm that, in fetal hemoglobin, the T in equilibrium R conformational equilibrium is more displaced toward the T conformation than in the adult form and indicate that increased electrostatic and hydrophobic protein-solvent interactions contribute to this effect. The data reported are discussed in terms of the known amino acid substitutions between the beta- and gamma-chains and an attempt is made to rationalize the results with a molecular mechanism based on the crystallographic structure of fetal deoxyhemoglobin.

Chemical Phenomena↗
PubMed1991

Protein dynamics. Comparative investigation on heme-proteins with different physiological roles.

We report the low temperature carbon monoxide recombination kinetics after photolysis and the temperature dependence of the visible absorption spectra of the isolated alpha SH-CO and beta SH-CO subunits from human hemoglobin A in ethylene glycol/water and in glycerol/water mixtures. Kinetic measurements on sperm whale (Physeter catodon) myoglobin and previously published optical spectroscopy data on the latter protein and on human hemoglobin A, in both solvents, (Cordone, L., A. Cupane, M. Leone, E. Vitrano, and D. Bulone. 1988. J. Mol. Biol. 199:312-218) are taken as reference. Low temperature flash photolysis data are analyzed within the multiple substates model proposed by Frauenfelder and co-workers (Austin, R. H., K. W. Beeson, L. Eisenstein, H. Frauenfelder, and I. C. Gunsalus. 1975. Biochemistry. 14:5355-5373). Within this model a distribution of activation enthalpies for ligand binding accounts for the structural heterogeneity of the protein, while the preexponential factor, containing also the entropic contribution to the free energy of the process, is considered to be constant for all conformational substates. Optical spectra are deconvoluted in gaussian components and the temperature dependence of the moments of the resulting bands is analyzed, within the harmonic Frank-Condon approximation, to obtain information on the stereodynamic properties of the heme pocket. The kinetic and spectral parameters thus obtained are found to be protein dependent also with respect to their sensitivity to changes in the composition of the external medium. A close correlation between the kinetic and spectral features is observed for the proteins examined under all experimental conditions studied. The results reported are discussed in terms of differences in the heme pocket structure and in the conformational heterogeneity among the various proteins, as related to their different capability to accommodate constraints imposed by the external medium.

Animals↗
PubMed1990

Oxygen binding to partially oxidized hemoglobin. Analysis in terms of an allosteric model.

We report on oxygen binding to partially oxidized (aquomet) hemoglobin. The fractional saturation with oxygen is evaluated by deconvoluting the optical absorption spectra, in the 500-700 nm wavelength region, in terms of oxyhemoglobin, deoxyhemoglobin and methemoglobin spectral components. Experiments have been performed with auto-oxidized samples and with samples obtained by mixing ferrous hemoglobin with fully oxidized hemoglobin (mixed samples). An increase in oxygen affinity and a decrease in cooperativity are observed on increasing the amount of ferric hemoglobin in the sample. A high cooperativity (nH approximately 2) is maintained even in the presence of 50-60% ferric hemes. Moreover, for equal amounts of methemoglobin the oxygen affinity is lower and the cooperativity higher for mixed samples than for those auto-oxidized. The results are analyzed within the framework of a modified Monod-Wyman-Changeux allosteric model taking into account the effects brought about by the presence of oxidized hemes and of alpha betta dimers. The distribution of ferric subunits within the tetramers in fully deoxygenated and fully oxygenated samples, as derived from the model, provides details on the cooperative behavior of partially oxidized hemoglobin.

Allosteric Regulation↗
PubMed1990

Optical absorption spectra of azurin and stellacyanin in glycerol/water and ethylene glycol/water solutions in the temperature range 290-20 K.

We have measured the optical absorption spectra of azurin and stellacyanin in the wavelength range 1100-350 nm and in the temperature interval 290-20 K. Samples are protein aqueous solutions containing 65% (v/v) glycerol or ethylene glycol as cryoprotectants and remain homogeneous and transparent throughout the whole temperature range investigated. Spectra are deconvoluted into Gaussian components and the temperature dependence of the zeroth, first and second moments of the various bands is analyzed, within the harmonic Franck-Condon approximation, to obtain information on the stereodynamic properties of the active sites of these proteins. Sizable differences of the integrated intensities of all the bands with temperature are observed and are attributed to variations of the metal-ligand relative positions (i.e., deformations of the active site) that occur as the temperature is lowered. The mean effective frequency of the nuclear vibrations coupled to all the observed bands is about 150 cm(-1) for both proteins in both solvents used; this indicates that the electronic transitions from which the optical spectrum originates are substantially coupled with low-frequency vibrational modes, likely ligand-metal-ligand deformations. The relevance of the stereodynamic properties of azurin and stellacyanin, investigated in this work, to their functional behavior is also suggested.

Journal Article↗
PubMed1988

Interaction between external medium and haem pocket in myoglobin probed by low-temperature optical spectroscopy.

The visible absorption spectra of carbonmonoxymyoglobin in the temperature range 300 to 20 K are reported and compared with the analogous spectra of carbonomonoxyhaemoglobin. The temperature dependence of the zeroth, first and second moment of the observed bands is analysed to obtain information on the local dynamics in the proximity of the haem. Contrary to haemoglobin, the first moment of the observed bands in myoglobin is markedly affected by the solvent composition and its value saturates at temperatures at which the solvent undergoes the glass transition. These data indicate that solvent properties influence the haem pocket stereodynamics in myoglobin; moreover, the different behaviour between myoglobin and haemoglobin suggests that the process should involve the surfaces that are buried in the haemoglobin tetramer and exposed to the solvent in myoglobin, and/or the different protein compressibility.

Animals↗
PubMed1986

The interaction of sodium maleate with human haemoglobin.

We have studied the effect of sodium maleate on the reaction of haemoglobin with oxygen at various temperatures between 10 degrees C and 30 degrees C. At all the temperatures investigated, the presence of sodium maleate causes a significant increase of log p50. Analysis of the experimental data in terms of Wyman's linkage theory indicates that one oxygen-linked maleate molecule bound to the haemoglobin tetramer in the deoxy conformation is released when the tetramer is oxygenated. Further analysis within the framework of the Monod-Wyman-Changeux model, considering maleate as a classical allosteric inhibitor, enabled us to obtain the equilibrium constant for the binding of maleate to deoxyhaemoglobin at various temperatures and therefore the values of the standard binding entropy and enthalpy. These last values indicate that the binding of maleate to deoxyhaemoglobin is an exothermic enthalpy-driven process.

Depression, Chemical↗
PubMed1986

Effect of organic co-solvents on the dimer/tetramer equilibrium of human haemoglobin.

We have studied the effect of organic co-solvents (monohydric alcohols and formamide) on the dimer-tetramer equilibrium of human haemoglobin by measuring the dependence of oxygen affinity upon haemoglobin concentration over a 100-fold concentration range and analysing the data with a modified Monod-Wyman-Changeux model in which the equilibrium (tetramer) in equilibrium with (dimer) + (dimer) was taken into account. This procedure enabled us to obtain the dimer-tetramer equilibrium constant and to find its dependence upon co-solvent concentration. Then by following the procedure already reported for the tense----relaxed state transition of haemoglobin, we separated the co-solvent effects into bulk electrostatic and non-bulk electrostatic (hydrophobic) contributions. We believe that our results demonstrate that during haemoglobin dissociation, just as we have already shown for the tense to relaxed state transition, both charged groups and hydrophobic surfaces became exposed to the solvent.

1-Propanol↗
PubMed1986

Effects of organic co-solvents on the oxygen equilibrium of N-ethylmaleimide-treated human haemoglobin.

We have studied the effects of monohydric alcohols and formamide on the oxygen equilibrium of native and N-ethylmaleimide-treated human haemoglobin. Comparison of the results obtained for the two haemoglobins gives further and compelling evidence in favour of the model, proposed recently by our group, on the role played by the solvent in the conformational equilibria of haemoglobin; moreover the results provide direct functional evidence of the relevance of the electrostatic free energy of salt bridges to the T in equilibrium with R equilibrium of haemoglobin.

1-Propanol↗