Molecular size of polymerized Hb negatively correlates with CD163 binding and Hb-induced HO-1 mRNA induction. (A) Four Oxyglobin fractions (fraction 1, 502 kDa; fraction 2, 272 kDa; fraction 3, 185 kDa; and fraction 4, 87 kDa) were isolated and purified using size-exclusion chromatography (left panel). Reanalysis of the purified fractions is shown in the right panel. (B) Each of the 4 oxyglobin fractions and native bovine Hb were tested for their ability to inhibit Hb-Hpfl (50 nM) uptake by CD163-HEK293 cells when applied in excess. Excess competitor concentrations were calculated by means of the absolute MW of the respective fraction. The increasing molecular size of each respective fraction is associated with diminished receptor interaction, as indicated by a decreased capacity to inhibit Hb-Hpfl uptake, even at high excess concentrations. ▴, fraction 1. •, fraction 2. (♦) fraction 3. ▾, fraction 4. ○, bovine Hb. (C) Heme oxygenase (HO-1) mRNA was measured after 8-hour incubation with each of the Oxyglobin fractions at concentrations of 1.5 μM (□) and 15 μM (▪). In accordance with limited CD163 interaction, HO-1 mRNA induction by the large (1 and 2) fractions of 502 kDa and 272 kDa was diminished. Data represent mean ± SD of 3 (B) and 2 (C) independent experiments.