Purification of human intrinsic factor by affinity chromatography

Abstract

Hydroxocobalamin was insolubilized by covalent coupling to albumin which in turn was coupled to bromoacetyl-activated cellulose. This product allowed specific adsorption of cobalamin-binding proteins from human gastric juice. The cobalamin-binding proteins were eluted by increase of temperature and addition of cyanocobalamin. After further chromatographic purification the net result of pure intrinsic factor was about 5% or some 75 mg from about 500 1 of gastric juice. The biological activity was proven by the Schilling test. It was homogeneous by sodium dodecyl sulphate gel electrophoresis and immunoelectrophoresis. Stokes radius (3.3 nm) was the same as the one of native intrinsic factor in gastric juice. The partial specific volume determined by ultracentrifugation in water and in deuterated solvent was 0.743 cm 3/g. The amino acid content was similar to the one of hog intrinsic factor. Sedimentation equilibrium ultracentrifugation indicated the presence of one or two protomers with a mol.wt of 30 000, unable to bind cyanocobalamin, and a monomer of mol.wt 60 000 binding one molecule of vitamin B 12.