Isolation of Vitamin B12-binding Proteins Using Affinity Chromatography: I. PREPARATION AND PROPERTIES OF VITAMIN B12-SEPHAROSE

Abstract

Abstract A method of affinity chromatography which is a potent tool for isolation of the trace vitamin B12 binding proteins has been developed. The affinity ligand was prepared by partial acid hydrolysis (0.4 n HCl, 64 hours, room temperature) of the amide groups of the unsubstituted propionamide side chains of the corrin ring of vitamin B12. The resultant mixture of mono-, di-, and tricarboxylic vitamin B12 derivatives was separated by chromatography on QAE-Sephadex. The monocarboxyl derivatives of vitamin B12 were coupled covalently to the free amino group of 3,3'-diaminodipropylamine-substituted Sepharose using 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide, thereby regenerating native vitamin B12 stably coupled to Sepharose (yield = 0.7 µmole of vitamin B12 per ml of packed Sepharose). The vitamin B12-Sepharose bound the vitamin B12 binding proteins (g90%) of human granulocytes, human plasma, human gastric juice, an extract of hog gastric mucosa, and a partially purified preparation of human transcobalamin II prepared from Cohn Fraction III of human plasma. The capacity of the vitamin B12 Sepharose to bind vitamin B12 binding proteins was at least 22% of the total vitamin B12 bound to Sepharose assuming 1 mole of vitamin B12 is bound per mole of vitamin B12 binding protein.