Figure 4.

sMD-2 binds heme. (A) To determine the physical interaction between sMD-2 and heme, human SS plasma was incubated overnight at 4°C with heme-agarose or control agarose beads. The pelleted beads were washed with PBS 6 times. The pull-down proteins bound to the beads were then run on an SDS-PAGE Western bot using anti-MD-2 IgG as the primary detection antibody. Six SS plasmas were examined in independent experiments with similar results. Representative Western blots of 2 SS human and mouse plasmas are shown. (B, C) UV/Vis absorption spectra (250 - 550 nm) of recombinant (B) sMD-2 and (C) hemopexin (HPX, positive control), with and without added heme. Absorbance spectra show scans of heme alone (black dashed line), recombinant protein alone (blue line) and recombinant protein plus heme (red line). The Soret peak at 414 nm, indicative of heme binding, increases in the presence of added heme (red) for sMD-2 and HPX. In the absence of added heme, both recombinant proteins appear to have some bound heme (blue line) as shown by the Soret peak at 414 nm. The UV/Vis scans (B, C) are representative of 3 independent experiments.