Figure 6
Characterization of heme- and receptor-binding by recombinant flagged wt-Hpx, ms-HpxHM and ms-HpxRM. Wt-Hpx-c-Flag, ms-HpxHM-c-Flag and ms-HpxRM-c-Flag plasmids were expressed in Chinese hamster ovary (CHO) cells and the recombinant Hpx proteins were purified from the CHO serum-free media 4 d after transfection by anti-flag affinity chromatography, as described in Materials and Methods. (A and B) Heme-binding of recombinant and purified rat Hpx was assessed by UV/Vis absorption spectrometry (250–600 nm). Heme bound to Hpx has a peak of absorbance at 414 nm. Wt-Hpx (A) and ms-HpxRM-Flag (B) can bind heme, but the ms-HpxHM-Flag (B) does not bind heme. (C) Binding of the recombinant flag Hpx proteins (1 µM) to HepG2 cells was assessed by immunofluorescence using and anti-flag IgG (Cy3/red) in the presence and absence of heme (1 µM). A 20-fold excess of purified human Hpx (20 µM) + wt-Hpx-Flag (1 µM) + heme (1 µM) was used to assess the specificity of binding (C, bottom panels). To determine co-localization of wt-Hpx + heme with CD91/LRP1, HepG2 cells were double immunostained for flag (Cy3/red) and CD91/LRP1 (FITC/green). White bar of scale equals 20 µM.