Isolation of vitamin B-12-binding proteins by combined immuno and affinity chromatography. Comparative studies on the isolated and unisolated proteins.
Intrinsic factor or cobalophilin were removed by incubating human gastric juice and pig pyloric extract with purified anti-intrinsic factor and anti-cobalophilin immunoglobulin-G, respectively, covalently coupled to Sepharose. Cobalophilin (transcobalamin I) was also removed from pig serum either by using anti-cobalophilin immunoglobulin-G Sepharose or by Sephadex G-200 chromatography. The one remaining semipurified vitamin B-12-binding protein (intrinsic factor, cobalophilin or transcobalamin II) was then isolated by vitamin B-12-Sepharose affinity chromatography. Intrinsic factors, cobalophilins and transcobalamin II isolated by this two-step procedure were compared by double isotope techniques with the corresponding protein not subjected to affinity chromatography and found to be identical in reaction to antiserum, gel filtration and electrofocusing. The avidity of the isolated and unisolated intrinsic factors for the ileal intrinsic factor receptor was also the same.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D005750:Gastric Juice / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D008970:Molecular Weight; D013045:Species Specificity; D013552:Swine; D014805:Vitamin B 12 / Q000378:metabolism*
D002352:Carrier Proteins; D014805:Vitamin B 12