The Vitamin B 12 -Binding Proteins of Some Amphibians

Abstract

Three immunologically distinct unsaturated (without vitamin) vitamin B₁₂-binding proteins have been demonstrated in humans. Intrinsic factor, the best known, has a molecular weight of 55,000 and promotes the uptake of vitamin B₁₂ by the intestinal epithelium (1, 2). Transcobalamin II, a liver-synthesized serum B12 binder with a molecular weight of 35,000, is apparently responsible for the uptake of the vitamin by the tissues (1, 2). A third binder, R binder or transcobalamin I, has a molecular weight of 110,000 and is found in serum, saliva, gastric juice, and a variety of other body fluids (1, 2). The function of this binder is not known.

It has been shown recently that chickens have a single unsaturated serum B¹² binder with a molecular weight of 113,000 and an immunologically similar proventriculus B₁₂ binder with a molecular weight of 96,000 (3). These binders superficially resemble the human R binders. They are approximately the same size as the human R binder, and in both cases immunologically similar forms occur in both the serum and gastric juice. However, antibody against the chicken binder does not react with human R binder (3). The present study demonstrates the presence of unsaturated B₁₂ binders in amphibians. These resemble neither the human binders nor the chicken binders.

Materials and Methods. Levels of unsaturated vitamin B₁₂ (B₁₂) binders were measured using zirconyl phosphate gel (Z-gel), a highly negatively charged gel that binds proteins below their isoelectric point (4, 5). B₁₂-⁵⁷Co was purchased from Philips-Duphar, Holland, and was diluted with unlabeled B₁₂ to give solutions containing either 100 ng/ml (1000 counts/ng) or 10 ng/ml (10,000 counts/ng). B₁₂-⁶⁰Co was used as purchased (1 μCi/1.484 μg or 0.652 μCi/μg; E. R. Squibb and Sons, New York).