Figure 3
From: Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis
Recombinant Fc-hHx has the same heme-binding activity as purified Hx. Recombinant Fc-hHx prepared as described in Materials and Methods was applied to SDS-PAGE with Coomassie Fluor Orange (Life Technologies) staining (A) and developed as a Western blot with Cy3-labeled goat anti-human Fc antibody (Jackson ImmunoResearch) (B). Fc-hHx is shown in the reduced gels as a high-purity 75-kDa monomer (see arrow). In nonreduced gels, the Fc-hHx is a 150-kDa dimer (not shown). (C) Heme binding assay was done with 10 µmol/L of recombinant Fc-hHx; purified hHx-B; Fc fragment of IgG1; human albumin (hALB). All protein samples or PBS were mixed with free heme at different concentrations (3 and 10 µm) at room temperature for 30 min. The absorbance at 413 nm was measured by Nanodrop ND1000. The results represent the mean ± SE.