Identification of recombinant Hpr as a hemoglobin-binding protein by hemoglobin-affinity chromatography and SPR analyses. (A) Silver-stained SDS-polyacrylamide gels of hemoglobin affinity-purified recombinant human Hp (phenotype 1-1, rHp1-1) and Hpr (rHpr). (B) SPR analysis of the binding of a range of concentrations of rHp1-1 and rHpr to immobilized hemoglobin A0 (Kd calculated to 2.3 nM and 13 nM, respectively). The slow dissociation (arrows, Kd < 2 × 10-4 s-1) indicates an almost irreversible binding. The concentration of rHp1-1 and rHpr is shown to the right of each curve. (C) SPR analysis of binding of rHpr or rHp1-1 to hemoglobin A0 in the presence of rHp1-1 and rHpr, respectively. The curves (solid lines) show binding of rHp1-1 (left panel) or rHpr (right panel) to the hemoglobin chip and subsequent binding of rHpr (arrow, left panel) and rHp1-1 (arrow, right panel). The dashed lines are transposed curves (from an identical chip), showing binding to the chip without prebound rHp1-1/rHpr, that is, the expected binding in the absence of competitive inhibitor. (D) SPR analysis of the binding of hemoglobin A0 (Hb), complexed with plasma-derived Hp1-1, recombinant rHp1-1, or rHpr, to immobilized CD163. The concentration of Hp1-1, rHp1-1, and rHpr is shown to the right of each curve.
