Photoaffinity labeling of the rat plasma vitamin D binding protein with [26,27-3H]-25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate].
It is well recognized that the vitamin D binding protein (DBP) is important for the transport of vitamin D, 25-hydroxyvitamin D (25-OH-D), and its metabolites. In an attempt to better understand the molecular-binding properties of this ubiquitous protein, we designed and synthesized a photoaffinity analogue of 25-OH-D3 and its radiolabeled counterpart. This analogue, 25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate] (25-OH-D3-ANG), was recognized by the rat DBP and was about 10 times less active than 25-OH-D3 in terms of binding. Incubation of [3H]25-OH-D3 or [3H]25-OH-D3-ANG with rat DBP revealed that both compounds were specifically bound to a protein with a sedimentation coefficient of 4.1 S. Each was displaced with a 500-fold excess of 25-OH-D3. When [3H]25-OH-D3-ANG was exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3, there was no displacement of tritium from the 4.1S peak. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of [3H]25-OH-D3-ANG exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3 revealed one major band with a molecular weight of 52 000. These data provide strong evidence that [3H]25-OH-D3-ANG was covalently linked to the rat DBP. This photoaffinity probe should provide a valuable tool for the analysis of the binding site on this transport protein.
D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism; D001667:Binding, Competitive; D002112:Calcifediol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; D007700:Kinetics; D051381:Rats; D014316:Tritium; D014809:Vitamin D-Binding Protein / Q000097:blood* / Q000302:isolation & purification
D000345:Affinity Labels; D001386:Azides; D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; D014809:Vitamin D-Binding Protein; D014316:Tritium; C050354:25-hydroxyvitamin D3 3-(N-(4-azido-2-nitrophenyl)glycinate); D002112:Calcifediol