Rapid purification of cobalamin-binding proteins using immobilized aminopropylcobalamin |
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Authors: | Donald W Jacobsen Yolanda D Montejano FM Huennekens |
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Institution: | Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037 USA |
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Abstract: | Aminopropylcobalamin (AP-Cbl), prepared from 3-chloropropylamine and cob(I)alamin, was immobilized on CNBr-activated Sephacryl beads. The product, Sephacryl-aminopropylcobalamin, contained ca. 1 μmol of AP-Cbl/ml of beads. Cobalamin-binding proteins in biological fluids were adsorbed selectively and quantitatively by Sephacryl-aminopropylcobalamin. After being washed to remove extraneous protein, the beads were photoirradiated to release the cobalamin-binding proteins as their aquacobalamin complexes. The latter could be converted to labeled cyanocobalamin complexes by treatment with 14C]KCN. The efficacy of this affinity chromatographic method is illustrated by the purification to near homogeneity and in high yield of three representative proteins: transcobalamin II from rabbit serum, intrinsic factor from human gastric juice, and R binder from human saliva. |
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