INTRINSIC-FACTOR COVALENTLY BOUND TO SEPHAROSE AS AFFINITY MEDIUM FORTHE PURIFICATION OF A SOLUBLE INTRINSIC-FACTOR RECEPTOR FROM HUMAN URINE

We have identified a soluble receptor for intrinsic factor (IF) in hum an urine. The purification of this protein by affinity chromatography required a preliminary purification of IF from hog pyloric mucosal ext ract. This was achieved by thermolabile cobalamin-ethanol-aminohexane Sepharose affinity chromatography with a 133-fold purification, a yiel d of 45% and a specific binding activity of 15720 pmol/mg protein. The purified Cbl-IF complex was coupled to epoxy-Sepharose with a yield o f 23.8% and a specific activity of 1.2 nmol per mol of gel. The solubl e IF receptor was purified form 200 mi of urine concentrate of pregnan t women. Desorption was performed at pH 5.0 and in the presence of 5 m M EDTA. The soluble IF receptor was purified 17 200-fold with a yield of 52% and a IF binding capacity of 3260 pmol per mg of protein. A sin gle protein with a M(r) of 70 000 was found in silver-stained SDS-PAGE .