37] Affinity chromatography of heme-binding proteins: synthesis of hemin-agarose

Abstract

This chapter describes the affinity chromatography of heme-binding proteins and the synthesis of hemin–agarose. Some features of the method for preparing hemin–agarose may be described as follows: (1) to avoid the solubility problem, the coupling reaction is performed in a pure nonaqueous solvent (100% DMF) in which hemin is highly soluble, (2) the immobilization is accomplished through the propionic acid group of hemin, (3) to activate the carboxyl group, 1, l ' -carbonyldiimidazole (CDI) is employed instead of more commonly used agents such as carbodiimides. This agent (CDI) is more convenient to use because there is no need to adjust the pH during the reaction, (4) a cross-linked agarose (Sepharose CL-6B) which is quite resistant to organic solvents and mechanical agitation is used as the matrix, (5) the time required to remove the unreacted hemin which is tightly adsorbed on the hemin–agarose is significantly reduced by introducing a wash with 25% pyridine, and (6) compared to other methods, a significantly higher coupling efficiency (1–2.5 μ mol hemin/ml agarose) is obtained. Some properties of the hemin–agarose resin prepared by the present synthetic procedure but using an uncross-linked agarose matrix have been described. Intactness of the ligand during the coupling reaction is demonstrated by the identical pyridine hemochrome absorption spectra of hemin and the hemin derivative released from the resin by alkali treatment. For affinity chromatography on the hemin–agarose, a high-ionic strength buffer (0.5 M NaCl and 10 mM sodium phosphate, pH 7.5) has been routinely used in the protein adsorption step to minimize nonspecific ionic interaction between protein and the affinity resin. The chapter also discusses the affinity chromatography of serum heme-binding proteins on hemin–agarose.