Hydrophobic interactions of the apo and holo forms of human cobalamin binding proteins

The interactions of transcobalamin II (TC II), intrinsic factor (IF) and R-type binding protein of cobalamin (Cb1, vitamin B₁₂) with the hydrophobic chromatography matrix Phenyl-Sepharose CL-4B were investigated. IF-Cb1 and R-Cb1 complexes were not adsorbed on Phenyl-Sepharose at room temperature or at 4°C with buffer containing 50 mM sodium phosphate, pH 7.4 containing 150 mM sodium chloride. The TC II-Cb1 complex adsorbed and could be eluted with buffer containing 50% $\text{v}\text{v}$ glycerol. IF without Cb1 adsorbed and was eluted with 50% glycerol at room temperature and 4°C. At room temperature, R binder without Cb1 eluted with buffer, but later than the R-Cb1 complex. At 4°C, R binder completely adsorbed to the matrix. TC II-without Cb1 bound to the matrix at 4°C and room temperature and could not be eluted with glycerol. These results suggest that Cb1 binding proteins can be separated and identified based on their hydrophobic properties. In addition, upon binding Cb1, TC II, IF and R-type binders undergo a conformational change such that the protein-Cb1 complex shows reduced hydrophobicity.