Gel filtration of vitamin B 12-binding materials from human gastric juice and gastric mucosa

Abstract

1. 1. In the course of experiments to isolate human intrinsic factor, its behavior on gel filtration through Sephadex G-100 at pH 7.3 was compared with that of the vitamin B 12-intrinsic factor complex from human gastric juice and gastric mucosa, and with bovine serum albumin and human hemoglobin. 2. 2. The vitamin B 12-binding materials of neutralized gastric juice showed the same elution pattern whether the vitamin B 12 was added before or after gel filtration. This pattern was the same as that obtained from gastric mucosal homogenates with added vitamin B 12. Two peaks of vitamin B 12-binding material were found, a small initial one comprising some 15–20% of the total vitamin B 12-binding activity of the specimen and a second peak representing 80–85% of the activity. 3. 3. The first peak gave no reaction in a specific radioimmunodiffusion assay for intrinsic factor. 4. 4. The second peak gave a strong reaction for intrinsic factor in the radioimmunodiffusion assay and its vitamin B 12-binding activity could be completely abolished by specific antibody, indicating that it was composed entirely or almost entirely of intrinsic factor. 5. 5. The intrinsic factor showed a distribution coefficient of 0.198 in this column, the same as bovine serum albumin. Since deviation from sphericity of the intrinsic factor and the presence of carbohydrate in the molecule would produce an overestimation of the molecular weight, it is probable that the molecular weight of the intrinsic factor has an upper limit of 65000–70000. There was no evidence for the presence of any intrinsic factor material of higher molecular weight than this either in the gastric mucosa or neutralized gastric juice. 6. 6. Addition of vitamin B 12 to the intrinsice factor did not alter its gel filtration behavior indicating that the binding of vitamin B 12 did not produce any gross changes in the molecule. 7. 7. The gel filtration with Sephadex G-100 gave a 10-fold purification of the intrinsic factor from gastric juice which was obtained in a yield of 80%.