AbstractThe adverse effects of extra-erythrocytic hemoglobin (Hb) are counterbalanced by several plasma proteins devoted to facilitate the clearance of free heme and Hb. In particular, haptoglobin (Hp) traps the αβ dimers of Hb, which are delivered to the reticulo-endothelial system by CD163 receptor-mediated endocytosis. Since Hp:Hb complexes show heme-based reactivity, kinetics of O₂ dissociation from the ferrous oxygenated human Hp1-1:Hb and Hp2-2:Hb complexes (Hp1-1:Hb(II)-O₂ and Hp2-2:Hb(II)-O₂, respectively) have been determined. O₂ dissociation from Hp1-1:Hb(II)-O₂ and Hp2-2:Hb(III)-O₂ follows a biphasic process. The relative amplitude of the fast and slow phases ranges between 0.47 and 0.53 of the total amplitude, with values of k_off1 (ranging between 25.6 ± 1.4 s⁻¹ and 29.1 ± 1.3 s⁻¹) being about twice faster than those of k_off2 (ranging between 13.8 ± 1.6 s⁻¹ and 16.1 ± 1.2 s⁻¹). Values of k_off1 and k_off2 are essentially the same independently on whether O₂ dissociation has been followed after addition of a dithionite solution or after O₂ displacement by a CO solution in the presence of dithionite. They correspond to those reported for the dissociation of the first O₂ molecule from tetrameric Hb(II)-O₂, indicating that in the R-state α and β chains are functionally heterogeneous and the tetramer and the dimer behave identically. Accordingly, the structural conformation of the α and β chains of the Hb dimer bound to Hp corresponds to that of the subunits of the Hb tetramer in the R-state.