Biochemical validation of HCAR2 as a receptor for heme, illustrated by direct ligand/receptor binding. (A) Comparison by SPR of the interaction of immobilized recombinant HCAR2 compared to C3b (negative control) with different concentrations of heme (39 to 2500 nM). (B) Analysis of HCAR2-heme interaction by spectroscopy (0.35 to 10.2 μM). (C) Absorbance at the Soret peak (414 nM) as a function of the molar excess of heme over the protein. RU, response units.