Calorimetric studies of inositol hexaphosphate binding to aquomethemoglobin A

Abstract

Inositol hexaphosphate (IHP) binding to aquomethemoglobin A (metHb) has been studied by flow microcalorimetry and compared to IHP interactions with carbonmonoxyhemoglobin A (COHb) and deoxyhemoglobin A (deoxyHb). At 25° and pH 6.8 in 0.05 M NaPhos containing 0.1 M NaCl and, after correction for heats of proton extraction from buffer, IHP binds to metHb, deoxyHb and COHb with enthalpies of −80.3, −104.2 and −105.9 kJ/mol IHP, respectively. None of the above processes exhibits large apparent heat capacity changes. The binding to metHb is strongly linked to proton uptake, 1.5 H + being absorbed per IHP bound at pH 6.8, with an enthalpic contribution of −21 kJ/mol H + absorbed. Inorganic anions, including chloride, phosphate and perchlorate, bind to metHb with dissociation constants in the 0.1 M range and are released upon IHP binding. At high salt concentrations 2–4 such ions are released with enthalpic contributions of +13 to +29 kJ/mol anion released. Thus observed heats of reaction represent a difference of large energetic terms, including not only intrinsic interaction of ligand with protein but also heats of proton extraction from buffer, proton uptake by protein and anion displacement by ligand, each of which can exceed 42 kJ/mol. These results are discussed in light of a possible allosteric transition accompanying IHP binding to metHbA.