Affinity Chromatography with 25-hydroxycholecalciferol Ester in the Isolation of the Binding Protein for Vitamin D and Its Metabolites from Human Serum

The 3 beta-hemisuccinate ester of 25-hydroxycholecalciferol was prepared by incubating the sterol with succinic acid anhydride in the presence of 4-dimethylaminopyridine at 4 degrees. The ester was isolated by silica gel column chromatography, and its hydrolysis in ethanolic sodium hydroxide yielded 25-hydroxycholecalciferol. The ester was covalently linked to aminoagarose in the presence of isobutylchloroformate, yielding 16 to 60 micrograms of sterol per packed ml of gel. Normal human serum was applied to the 3 beta-hemisuccinate-25-OH-D3 aminoagarose gel and elutions with buffer of increasing molarity were carried out. Total protein and DBP content of the 6M guanidine elution fractions revealed a 12 to 134-fold purification of human serum DBP by this technique. Best purifications were achieved by incubating serum with the gel at pH 8.3 and rinsing the gel with 0.3 M KCl prior to its elution with the guanidine. Our results indicate that affinity chromatography with this ligand, or possibly other vitamin D sterol esters, is a practical and useful technique in the analysis and preparative isolation of vitamin D sterol-binding proteins.