A new method for affinity chromatography of heme-binding proteins: Synthesis and characterization of hematin- and hematoporphyrin-agarose

Abstract

Two new affinity columns, using hematin and hematoporphyrin as ligands, have been prepared. Both were made by first attaching 1,6-diaminohexane to Sepharose 4B by the cyanogen bromide procedure and then coupling the porphyrins to the free amino groups of this arm with carbodiimide. This second reaction was done in dimethylformamide to increase the solubility of the porphyrins at pH 4.7. This resin was then washed extensively with dimethylformamide to remove all of the unreacted porphyrin. The new affinity columns are able to bind apoglobin, albumin, and hemopexin, which demonstrates their ability to purify heme-binding proteins. The proteins could be removed by washing the column with a deforming buffer, such as 8 m urea or 0.1 m sodium citrate buffer at pH 4. Neither lysozyme nor hemoglobin bound to these resins demonstrating that the absorbants are specific for apo heme-binding proteins.