Nature of vitamin B 12 binding : II. Steric orientation of vitamin B 12 on binding and number of combining sites of human intrinsic factor and the transcobalamins

Abstract

The steric orientation of vitamin B 12 on binding to human intrinsic factor, transcobalamin 1 and transcobalamin II has been studied by use of different cobalamin derivatives. Covalent coupling of a protein to 5′-deoxyadenosylcobalamin via the deoxyadenosyl group or to hydroxocobalamin via the phosphate group does not prevent binding to intrinsic factor, transcobalamin I or transcobalamin II. Dicyanocobinamide competes with the binding of cyanocobalamin. Structural changes in the C pyrrol ring of the corrin moiety (anilide of hydroxocobalamin) do not inhibit B 12 binding while changes in the B pyrrol ring (lactone of cyanocabolamin) do. The benzimidazole ring in the nucleotide also appears to be involved in the binding process. It is concluded that the area of contact for intrinsic factor, transcobalamin I and transcobalamin II is in the region of the edge of the B ring in the corrin, probably with overlap to the adjoining part of the dimethylbenzimidazole. Measurements of Stokes radius of the binders, trace labelled with [ 57Co]vitamin B 12 and mixed with hydroxocobalamin coupled to albumin, reveals that each of the three binders only has one combining site.