Calorimetric Studies of Hemoglobin Function, the Binding of 2,3-Diphosphoglycerate and Inositol Hexaphosphate to Human Hemoglobin A

Abstract

Abstract Calorimetric titration curves demonstrate the heat of binding of organic phosphates to oxy- and deoxyhemoglobin at pH 7.4 in 0.05 m 2,2-bis(hydroxymethyl)-2,2',2''-nitrilo-ethanol buffer and 0.1 m Cl-. An absence of binding heat was noted at this pH value for both the 2,3-diphosphoglycerate (2,3-DPG) and inositol hexaphosphate (IHP) interactions with oxyhemoglobin. At pH 6.0, an exothermic heat of reaction is observed between both oxy- and deoxyhemoglobin and 2,3-DPG, the deoxy complex being the more stable. The binding ratio of 2,3-DPG and IHP to deoxyhemoglobin at all pH values studied was found to be 1:1. A strong pH dependence in the ΔH for these reactions suggests a proton involvement. The pH coefficient of binding is markedly different between the two forms of reduced hemoglobin. Mg2+ is shown to be a competitive inhibitor of 2,3-DPG-deoxyhemoglobin binding through formation of a nonbinding 2,3-DPG-Mg2+ complex. IHP forms a more stable complex than 2,3-DPG with deoxyhemoglobin at pH 7.0 to 7.4, and it shows reactivity at pH 7.4 with methemoglobin. This complex seems to be quite stable with a small heat of formation and involves all hemoglobin molecules, not just those in a particular spin state. 2,3-DPG, on the other hand, gives little or no heat of binding to methemoglobin at pH 7.4 but shows appreciable binding at pH 6.0. Differences in binding heats between the three hemoglobin forms are interpreted to mean that different functional groups participate in the binding of the same ligand to the various hemoglobin quaternary states.