The functional properties of dromedary hemoglobin have been studied as a function of chloride, polyphosphates and pH and compared with those of human hemoglobin. The two proteins have the same amino acid residues at the anion-binding sites as well as at the level of the groups responsible for the alkaline Bohr effect. Analysis of the experimental data reveals that: (a) intrinsic oxygen affinity and the Bohr effect are very similar for the two proteins; (b) the association equilibrium constants of chloride are substantially higher in the dromedary system, both in the unligated and ligated state; (c) two polyanion-binding sites occur in dromedary oxy and deoxyhemoglobin; (d) association constants of polyphosphates for the higher-affinity binding site (probably in the cavity between p chains) are comparable for the two proteins under physiological conditions; association constants for the second binding site in dromedary hemoglobin are not affected by pH changes; (e) the dependence of oxygen affinity in dromedary hemoglobin upon chloride concentration is complex, this anion at relatively low concentrations reverses the effect of millimolar polyphosphate; (0 both in stopped-flow and flash photolysis experiments the kinetic behaviour of dromedary hemoglobin is consistent with the equilibrium results.The pronounced sensitivity to solvent composition of the functional properties of dromedary hemoglobin even in the oxy state stresses the potential relevance of this conformation for regulating the oxygen transport in vivo.Dromedary (Camelus dromedarius) can live without water for long periods of time, losing about 30% of its body weight. A dehydrated dromedary can regain its losses of water (more than 100 1) in about 10 min so that rapid modifications in blood composition are determined. The adaptations of the oval-shaped erythrocytes of dromedary to dehydration and rehydration [I -41 result in changes of cell dimensions (in addition to the modification of other parameters, such as the lifespan and shape). This evidence suggests that dromedary hemoglobin is exposed to a wide variety of chemical stimuli due to the extremely variable environmental conditions. Since the primary function of hemoglobin, oxygen transport, is usually modulated by the intra-erythrocyte components, dromedary hemoglobin may be expected to show some peculiar heterotropic properties. It is well known, in fact, that some animals have hemoglobins which seem to be totally unaffected by variation in cellular components, whereas the hemoglobins of others are very sensitive to even small changes in the environment [5, 61. Therefore, in order to gain a better insight on how dromedary hemoglobin function is affected by the solvent components to meet the changing needs of the animal, the influence of some enivronmental parameters on heme ligand binding was investigated. Studies on human hemoglobin have been included for comparative purposes.The primary structure of hemoglobin from Camelus dromrdarius has now been established Abbreviations. Glycerate-2,3-P2 o...
The energetics of signal propagation between different functional domains (i.e. the binding sites for O 2 , inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA 0 was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to ؊3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O 2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO 2 ؍ 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T-and R-like tertiary conformation(s) within a quaternary R-like structure).Binding properties of gaseous ligands to human HbA 0 1 are markedly influenced by the so-called third components (in addition to HbA 0 and heme's ligands), such as organic phosphates (e.g. 2,3-bisphosphoglycerate and inositol hexakisphosphate (IHP)), protons, and chloride ions (1-4), which interact with HbA 0 at sites topologically different (heterotropic sites) from the heme.These heterotropic functional effects have been previously investigated in thermodynamic (5, 6) and kinetic (7) terms, leading to the evidence of a network of interplays among different sites, such that linkage relationships can be put in evidence between different heterotropic ligands. However, a quantitative investigation of these effects has never been performed since the information is usually obtained through a differential influence of the non-heme binding properties for the deoxygenated and oxygenated derivatives of HbA 0 .We have recently reported on the IHP binding properties to some heme ligated forms of ferrous HbA 0 in the presence of 0.1 M chloride, showing the existence of two binding sites for IHP with a similar affinity independently of the heme ligand (8). In addition, a recent thermodynamic study has been carried out on the interaction of IHP with oxygenated HbA 0 (9), leading to the evidence that the inter-relationship between protons and IHP is regulated by the pK a shift of three classes of residues in HbA 0 , and it is characterized by a proton-linked balancing between the enthalpic and entropic contribution. Furthermore, in the last few years particular interest has been addressed toward the effect of bezafibrate (BZF) (see Fig. 1) on the O 2 -binding properties of HbA 0 , and mostly on the original observation that its effect is potentiated by the presence of organic phosphates (10, 11), indicating that the two heterotropic ligands bind to different sites (12). This observation is interesting, since it underlies the existence...
The c.d. spectrum of oxyhaemoglobin from Camelus dromedarius is significantly affected by the presence of inositol hexakisphosphate. Correlation with O2-binding measurements shows that these dichroic changes parallel the functional properties of the protein. The optical modifications suggest that, in contrast with human haemoglobin, the conformational changes induced by inositol hexakisphosphate on dromedary oxyhaemoglobin are mainly attributable to a local change of the tertiary structure reminiscent of that of the deoxy derivative, the quaternary conformation seeming to be almost unaffected. The results provide direct evidence of the existence on the protein of two distinct sites for polyanions.
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