Recently I am working on expression and purification of mammalian cytochrome b5, a heme protein with the molecular weight of ~10 KDa. Because of its low heme incorporation in recombinant expression, this protein was reconstituted by adding free hemin followed by Ni-NTA purification. Once the restituted protein mixture was loaded onto the column, the resins were dark/greenish, reflecting the presence of free hemin. In the wash step (Kpi buffer + 10 mM imidazole), most of resins turned to dark red. I thought that this dark red stuff would be the reconstituted heme protein. But in the elution step, most of red stuff cannot come off using up to 500 mM imidazole (pH 8.0).
So I wonder whether this red color is due to a chemical reaction between free hemin and nickel-NTA? Any suggestions are welcome.