Purification and molecular characterization of human transcobalamin II.
Transcobalamin II (TCII) has been purified from Cohn fraction III of human plasma by batchwise binding to and then elution from carboxymethyl-Sephadex, affinity chromatography using photo-labile aminopropyl cobalamin coupled to activated Sephacryl S-200, and finally chromatography through carboxymethyl cellulose. The yield was approximately 80%. The addition of protease inhibitors in all steps of the purification procedure and extensive washing of the carboxymethyl-Sephadex prior to eluting the TCII minimized degradation of the protein and the final preparation of holo-TCII contained 1 mol of cobalamin/mol of protein. A single polypeptide of 43,000 daltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 19 amino acids have been determined for human TCII. 12 of the amino acids are homologous with rabbit TCII and six are homologous with human R-binder, but there is no homology with human intrinsic factor.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007122:Immunoelectrophoresis; D008970:Molecular Weight; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011817:Rabbits; D013053:Spectrophotometry; D014155:Transcobalamins / Q000302:isolation & purification*; D014805:Vitamin B 12 / Q000032:analysis
D000596:Amino Acids; D010446:Peptide Fragments; D011480:Protease Inhibitors; D014155:Transcobalamins; D014805:Vitamin B 12