Isolation and partial characterization of a human vitamin D-binding plasma protein.

Vitamin D circulates in human plasma bound to a specific transport protein. This protein differs from the lipoproteins and has a hydrated density greater than 1.21. The purification of the human vitamin D-binding protein was accomplished by use of ammonium sulfate fractionation, DEAE-Sephadex chromatography, sulfoethyl-Sephadex chromatography, and gel chromatography. These procedures resulted in a highly purified preparation of the vitamin D-binding protein which had been pfirified approximately 15,000-fold. The purified protein appeared homogeneous by Ouchterlony immunodiffusion analyses, immunoelectrophoresis, and by analytical ultracentrifugation. The vitamin D-binding protein separated into two components on electrophoresis, both with 01~ mobility. The most anodal component carried vitamin Da, whereas the cathodal form of the vitamin D-binding protein was devoid of this form of the vitamin. The molecular weight of the vitamin D-binding protein determined by equilibrium ultracentrifugation and estimated from the sedimentation coefficient and gel chromatography was approximately 53,000. Determinations of the molecular weight of reduced and alkylated vitamin Dbinding protein in 6 M guanidine hydrochloride gave the same value as found under physiological conditions, suggesting that this protein is not composed of subunits. The frictional ratio (f/f,,) was low for the vitamin D-binding protein, indicating a close to spherical appearance for this protein. The occurrence of the vitamin D-binding protein in normal serum, normal urine, and normal cerebrospinal fluid was established by Ouchterlony immunodiffusion analyses with use of a specific antiserum against the vitamin D-binding protein. Indirect estimates indicated that the normal concentration of this protein in serum is approximately 5 pg per ml.