An improved method for large scale purification of human holo-transcobalamin II.

25 mg of human holo-transcobalamin II with a specific cobalamin-binding capacity of 0.95 mol cobalamin/mol TC II was purified from 122 kg Cohn fraction III with a yield of 73% and a purification factor of 9.34 . 10(5). Consecutive purification steps comprised CM-Sephadex batchwise ion-exchange chromatography, affinity chromatography, using cyanocobalamin as a ligand, thermolability attached to 3.3'-diaminodipropylamine-substituted CH-Sepharose, and gel filtration. The high yield of the purification procedure was achieved by improving the stability of apo-transcobalamin II in the eluate of the CM-Sephadex, and by a few other modifications of a former procedure. In the latter, rapid denaturation of apo-transcobalamin II prohibited the use of long term affinity chromatography, which is obligatory for processing large amounts of Crohn fraction. In addition, subfractionation of transcobalamin II into smaller fragments which occurred in SDS-polyacrylamide gel electrophoresis in previous studies, was now reduced, indicating that proteolysis in the CM-Sephadex eluate had been prevented effectively.