Isolation of vitamin B12-binding proteins using affinity chromatography. I. Preparation and properties of vitamin B12-sepharose.

A method of affinity chromatography which is a potent tool for isolation of the trace vitamin Blz 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 Blz. The resultant mixture of mono-, di-, and tricarboxylic vitamin BN derivatives was separated by chromatography on QAE-Sephadex. The monocarboxyl derivatives of vitamin Blz were coupled covalently to the free amino group of 3,3’-diaminodipropylamine-substituted Sepharose using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, thereby regenerating native vitamin Blz stably coupled to Sepharose (yield = 0.7 itmole of vitamin Blz per ml of packed Sepharose). The vitamin B&epharose bound the vitamin Blz binding proteins ( >QO %) 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 Blz Sepharose to bind vitamin Blz binding proteins was at least 22% of the total vitamin Blz bound to Sepharose assuming 1 mole of vitamin Blz is bound per mole of vitamin Biz binding protein.