Thermodynamics of Inositol Hexakisphosphate Interaction with Human Oxyhemoglobin

Abstract

, which are located in the ␤ 1 ␤ 2 dyad axis, where they have been also proposed to interact with 2,3-diphosphoglycerate, whereas the third group does not appear easily identifiable. Calorimetric measurements of the heat associated with IHP binding at different pH values over the same range indicate that IHP binding is mostly enthalpy-driven at pH < 7 and mostly entropydriven at pH > 7.Human hemoglobin (Hb) 1 is functionally modulated by several non-heme ligands, such as organic phosphates (i.e. 2,3-diphosphoglycerate (2,3-DPG) and myo-inositol hexakisphosphate (IHP)), protons, and chloride ions (1-5), which bind at heterotropic interaction sites, topologically distinct from the heme at which homotropic ligands bind.The structure of this binding pocket has been determined for the interaction of 2,3-DPG (6), which has been shown to bind at the interface between the two ␤-chains, mainly involving three residues from either one of ␤-subunits (i.e. HisNA2(␤2), Lys-EF6(␤82), and HisH21(␤143), see Ref. 6).In more recent years, another organic phosphate, namely IHP (closely related to the inositol pentaphosphate, which is the physiological effector in avian erythrocytes; see Ref. 7), has often been employed to study the modulation of functional properties of human Hb (8, 9). It possesses additional negative charges with respect to 2,3-DPG, and it displays a much larger effect, which suggests the occurrence of additional electrostatic interactions with respect to 2,3-DPG, as from early model building studies on deoxy Hb (10). Therefore, the enhanced functional effect of IHP on the O 2 binding properties of human Hb with respect to 2,3-DPG could be related to a more widespread interaction surface, with the possibility of modulating ligand-linked conformational changes taking place over a larger portion of the whole tetramer.However, a comprehension of the origin for this enhanced effect starts from the characterization of the IHP interaction energy with deoxyHb and with oxyHb. Previous studies have shown that IHP binds HbO 2 , and its binding properties are pH-dependent (11,12). In this study, we have carried out a detailed analysis of the interaction of IHP with human HbO 2 , measuring the effect on (a) proton titration, (b) O 2 dissociation kinetics from fully liganded tetramer, and (c) heat associated to the reaction in order to give a quantitative description of the system and of the interplay between IHP and proton interaction with human HbO 2 . EXPERIMENTAL PROCEDURESHuman HbO 2 was obtained from the blood of healthy volunteers and stripped of anions according to the procedure reported by Riggs (13). Cells were washed three times with iso-osmotic NaCl solutions by centrifugation at 1000 ϫ g, and packed cells were lysed by adding 2 volumes of cold bidistilled water. Stroma were removed by centrifugation at 12,000 ϫ g for 30 min. Hemolysate was first filtered through a Sephadex G-25 column, equilibrated with 0.01 M Tris/HCl buffer, pH 8.0, and EDTA 10 Ϫ5 M, and afterward it was passed through a column of mixed bed ...

Structural and functional analysis of the two haemoglobins of the Antarctic seabird Catharacta maccormicki

“…All phosphate groups were considered to be completely charged. In fact, the InsP 6 charge in solution, at pH 7.5, is < 28 [19], whereas when it is bound to Hb the pK a values of its phosphate groups decrease [20,21]. The InsP 6 model obtained was identical to that reported by Arnone and Perutz [20] in the crystallographic structure of the deoxy-Hb A±InsP 6 complex.…”

“…6. The results obtained at different temperatures were used to calculate the DH values which, expressed as kcal´mol 21 oxygen, were 210.4 and 210.2 for K T , and 210.6 and 211.1 for K R , for Hb 1 and Hb , respectively. These data suggest that temperature affects the binding of oxygen to the first and to the fourth site to a similar extent in the two Hbs.…”

“…The eluents were (A) 40% acetonitrile containing 0.3% trifluoroacetic acid and (B) 60% acetonitrile. Flow rate was 1 mL´min 21 . Cellulose acetate electrophoresis of haemolysates and purified Hbs, as well as SDS/PAGE of purified globins, were carried out as described [6,7].…”

Mammalian Phosphomannomutase PMM1 Is the Brain IMP-sensitive Glucose-1,6-bisphosphatase

“…Alternatively, free Glc-1,6-P 2 may represent only a small fraction of total Glc-1,6-P 2 in erythrocytes due to binding to hemoglobin. Hemoglobin is indeed known to bind avidly multiply charged phosphate esters such as 2,3-bisphosphoglycerate and inositol pentakisphosphate (33). It is therefore likely that it will also bind with great affinity bisphosphate esters such as Glc-1,6-P 2 .…”

Membrane Recognition by Vesicular Stomatitis Virus Involves Enthalpy-Driven Protein-Lipid Interactions

“…ITC is the only technique that allows the direct thermodynamic analysis of biomolecular interactions, providing the binding constant and stoichiometry in addition to the enthalpy and entropy of binding. Several ITC studies showed that electrostatic binding is usually driven by enthalpy (3,26,31,41,50). However, it is important to point out that the calorimetric enthalpy is actually a sum of all the heat effects, endothermic and exothermic, taking place during the interaction.…”