FIG. 2.

Additional evidence for heme transfer from holo-Shp to apo-HtsA. Apo-HtsA (10.5 μM) was incubated with 13 μM holo-Shp in 0.5 ml of 20 mM Tris-HCl, pH 8.0, at room temperature for 2 h. The sample was then loaded onto a column with 0.2 ml Ni-NTA resin and eluted as described in the text. (A) SDS-PAGE analysis showing separation of HtsA from its mixture with Shp. Lanes: 1, control HtsA; 2, the incubated mixture of apo-HtsA and holo-Shp before separation; 3, HtsA isolated from the mixture using Ni-NTA; 4, control Shp. (B) The spectra of apo-HtsA (dashed curves) and HtsA (solid curve) isolated from the holo-Shp/apo-HtsA mixture. (C) Photograph showing the yellow band of the following samples resolved by native PAGE. Lanes: 1, reconstituted holo-HtsA; 2, HtsA isolated from the holo-Shp/apo-HtsA mixture.