36] Affinity chromatography of heme-binding proteins: synthesis and characterization of hematin- and hematoporphyrin-agarose

Abstract

This chapter discusses a method for making a hematin–agarose resin that has been used to successfully purify hemopexin. The method can be used to synthesize any porphyrin–agarose column packing. First, aminohexyl–agarose is made using the cyanogen bromide method, and then the porphyrin is attached with a carbodiimide reaction. For the hematin–agarose, it is critical that the second reaction be done in dimethylformamide so that the hematin is soluble. The synthesis of porphyrin–agarose resin is a two-step procedure. In the first phase the agarose is activated by cyanogen bromide, and then coupled to 1,6-diaminohexane. In a typical synthesis, well-washed Sepharose 4B (150 ml) was activated by cyanogen bromide (37.5 g) at pH 10.5 and 25°. In the second phase of the synthesis, the porphyrin is linked to the free amino group of the aminohexyl “arm” by a carbodiimide reaction. The amount of porphyrin attached to the resin is determined spectrophotometrically. Standard curves are constructed by adding known amounts of hematin or hematoporphyrin to the same volume of suspended agarose. The hematin–agarose resin can be used to further purify hemopexin from a crude preparation. To demonstrate that the columns are specific for heme-binding proteins, lysozyme and hemoglobin are applied to hematin–agarose. In the synthesis of the porphyrin affinity resins, the aminohexylagarose is first synthesized by the cyanogen bromide technique and then hematin is coupled to the “arm” by the carbodiimide reaction. The porphyrin in the affinity resin can be attached by either or both of its propionic acid groups. The novel and essential aspect of this reaction is the use of pure DMF as a solvent for hematin.