Alteration of human intrinsic factor during affinity chromatography purification using concentrated guanidine

Affinity chromatography using vitamin Bra (Bra) covalently coupled to Sepharose has been used to isolate from solution a number of Bra-binding proteins including gastric intrinsic factor (IF) [ 1,2] . The procedure requires the use of concentrated guanidineHCl, a known protein denaturant, to dissociate the binding protein from the Bra-Sepharose complex. The Bra-binding proteins, like other ligand-binding transport macromolecules, are structurally bifunctional; that is, there are two biologically important reacting sites, one which binds the ligand-substrate and the second which attaches to a receptor site on the cell. When such a binding protein is isolated by affinity chromatography using the ligandsubstrate coupled to the solid matrix, the ligand binding site on the macromolecule becomes structurally fured, whereas the receptor binding site remains exposed and more susceptible to chemical alteration. This report will present evidence that human gastric intrinsic factor purified by elution from Bra-Sepharose column by 7.5 M guanidine-HCl, although structurally homogeneous by disc-gel electrophoresis, is nevertheless different than the native IF in gastric juice because there is decreased affinity for the intestinal receptor.