25-Hydroxyvitamin D Transport in Human Plasma. Isolation and Partial Characterization of Calcifidiol-binding Protein

The binding protein for 25-hydroxycholecalciferol (25-OH-D3 or calcifidiol) in human plasma has been purified from Cohn Fraction IV. Following in vitro labeling with 25-OH-[3H]D3, the isolation sequence of procedures included: DEAE-cellulose chromatography; gel filtration on Sephadex G-200; chromatography on DEAE-Sephadex; preparative polyacrylamide gel electrophoresis. These procedures resulted in a calcifidiol-binding protein (Cal-BP) which had been purified approximately 170-fold, and which was homogeneous by physical and immunological criteria. The purified Cal-BP had inter-alpha mobility, a sedimentation constant (S20, w) of 3.46 S, and a molecular weight of approximately 59,000. Sucrose gradient ultracentrifugation of Cal-BP and ligand incubations indicated that there was apparently one binding site for either cholecalciferol, calcifidiol or 1,25-dihydroxycholecalciferol per molecule of human Cal-BP. The protein had highest affinity for calcifidiol, displaying an apparent dissociation constant (Kd) of 6.4 X 10(-8) M for this sterol. Specific anti-human Cal-BP antisera were prepared in rabbits, and produced precipitate lines of identity between Cal-BP and human serum. Specific binding of vitamin D3,25-OH-D3, and 1,25-(OH)2D3 by human serum was completely neutralized after immunoprecipitation of the serum with the gamma globulin fraction of anti-Cal-BP antiserum, indicating a common transport protein for these sterols in human plasma. There was no immunological cross-reactivity between Cal-BP and rat or chicken sera, indicating that the Cal-BP in these three sera are immunologically completely distinct. Purified human Cal-BP and human sera also produced lines of identity with commercial anti-human group-specific component (Gc) antisera in radial immunodiffusion experiments. This finding supports an earlier report of the identity of calciferol/calcifidiol-binding protein and group-specific component in human serum. The Cal-BP content of human serum is approximately 10(-5) M, whereas the calcifidiol content is approximately 10(-7) M. Normally, the dominant moiety of human plasma Cal-BP is the apoprotein.