Isolation and Partial Characterization of a Human Vitamin D-binding Plasma Protein

Abstract

Abstract 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 purified 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 α1 mobility. The most anodal component carried vitamin D3, 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 D-binding 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/f0) 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 µg per ml.