Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3.
3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3.
D000345:Affinity Labels / Q000138:chemical synthesis / Q000494:pharmacology*; D001386:Azides / Q000138:chemical synthesis / Q000494:pharmacology* / Q000528:radiation effects; D001665:Binding Sites; D002112:Calcifediol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology / Q000528:radiation effects; D004355:Drug Stability; D004586:Electrophoresis; D005470:Fluorometry / Q000379:methods; D006801:Humans; D007700:Kinetics; D013997:Time Factors; D014316:Tritium; D014466:Ultraviolet Rays; D014809:Vitamin D-Binding Protein / Q000097:blood / Q000378:metabolism* / Q000528:radiation effects
D000345:Affinity Labels; D001386:Azides; D014809:Vitamin D-Binding Protein; D014316:Tritium; C053859:3-deoxy-3-azido-25-hydroxyvitamin D3; D002112:Calcifediol