[4] Purification of B12-binding proteins using a photodissociative affinity matrix

Publisher Summary This chapter outlines a general method for the affinity purification of Cbl-binding proteins from body fluids or crude extracts utilizing a photodissociative Cbl matrix. B 12 -binding proteins I play essential roles in the absorption, distribution, and retention of vitamin B 12 (cyanocobalamin; CN-Cbl) and its coenzyme forms, adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl).The unique organometallic carbon–cobalt bond of alkylcorrinoids homolyzes when exposed to light in the UV-visible range. In most alkylcorrinoids this bond is located in the β or upper-axial ligand position. Analogs containing aminoalkyl groups in the β position readily react with activated solid-phase matrices to give immobilized corrinoids that retain their high avidity for apoCbl-binding proteins. When exposed to visible light, the organometallic bond photodissociates and releases an aquacorrinoid-protein complex to solution. The chapter discusses the preparation of photodissociative corrinoid matrices. The affinity purification of Cbl-binding proteins involves: the preparation of body fluids and tissue extracts, adsorption of apoCbl-binding proteins by S-APCbl, and photodissociation of Cbl-binding proteins from S-APCbl.