Monocarboxylic acid derivatives of vitamin B12 were covalently coupled to 1,6-hexanediamine-substituted Sepharose by using a water-soluble carbodiimide resulting in 1.32 umoles of B12 coupled per ml of Sepharose. After a source of crude hog intrinsic factor (IF) was passed over the column, a selective linear gradient of guanidine HCl (0 to 4.0 M) was used to remove IF and 4.0 to 7.5 M to elute NIF (a vitamin B12-binding glycoprotein not active in promoting vitamin B12 absorption). Anti-IF antibodies blocked 99% of the B12 binding by the isolated IF and only 1% of the B12 binding by NIF. Passage over a hydroxyapatite column resulted in IF 99% pure with a specific activity of 29.8 μg of B12 binding per mg of protein. IF so isolated exhibited one homogeneous band on polyacrylamide gel electrophoresis and corrected B12 malabsorption in a patient with pernicious anemia.
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