Fluoride and azide binding to ferric human hemoglobin:haptoglobin complexes highlights the ligand-dependent inequivalence of the α and ß hemoglobin chains.

ABSTRACT

Haptoglobin (Hp) binds human hemoglobin (Hb), contributing to prevent extra-erythrocytic Hb-induced damage. Hp forms preferentially complexes with αß dimers, displaying heme-based reactivity. Here, kinetics and thermodynamics of fluoride and azide binding to ferric human Hb (Hb(III)) complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1Hb(III) and Hp2-2Hb(III), respectively) are reported (pH 7.0 and 20.0 °C). Fluoride binds to Hp1-1Hb(III) and Hp2-2Hb(III) with a one-step kinetic and equilibrium behavior. In contrast, kinetics of azide binding to and dissociation from Hp1-1Hb(III)(-N3-) and Hp2-2Hb(III)(-N3-) follow a two-step process. However, azide binding to Hp1-1Hb(III) and Hp2-2Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide binding to Hp1-1Hb(III) and Hp2-2Hb(III) should reflect azide binding to the ferric ß and α chains, respectively, as also proposed for the similar behavior observed in Hb(III). Present results highlight the ligand-dependent kinetic inequivalence of Hb subunits in the ferric form, reflecting structural differences between the two subunits in the interaction with some ferric ligands.