Purification of hog gastric intrinsic factor by a simple two-step procedure based on affinity chromatography and a selective guanidine hydrochloride gradient.
Monocarboxylic acid derivaties of vitamin B12 were covalently coupled to 1,6-hexanediamine-substituted Sepharose by using a water-soluble carbodiimide resulting in 1.32 micronmoles 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 HC1 (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 microng 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.
D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005753:Gastric Mucosa / Q000032:analysis; D006146:Guanidines; D006882:Hydroxyapatites; D007437:Intrinsic Factor / Q000302:isolation & purification*; D008722:Methods; D013552:Swine; D014805:Vitamin B 12
D006146:Guanidines; D006882:Hydroxyapatites; D007437:Intrinsic Factor; D014805:Vitamin B 12