Purification of vitamin D binding protein from human plasma using high performance liquid chromatography

Abstract

Vitamin D binding protein/group-specific component was purified from human plasma by chromatographic techniques utilising high performance liquid chromatography and by traditional low pressure Chromatographic techniques alone. Use of high performance liquid chromatography considerably reduced the time taken to prepare pure vitamin D binding protein and increased the yield to 16% compared with 2.8% using the traditional methods. The vitamin D binding protein prepared by high performance liquid chromatography was shown to be highly pure by amino acid sequence, SDS gel electrophoresis and by antibody production. The amino acid sequence was confirmed and extended. The affinity constants of the high pressure liquid chromatography purified vitamin D binding protein for 25 hydroxycholecalciferol (25 OHD 3) and 1,25 dihydroxycholecalciferol 1,25(OH) 2D 3 were 1.9 × 10 7 mol/l and 2.6 × 10 6 mol/l, respectively.