25-Hydroxy[26,27-methyl-3H]vitamin D3-3 beta-(1,2-epoxypropyl)ether: an affinity labeling reagent for human vitamin...

Vitamin D-binding protein (DBP) is primarily involved in the binding and transportation of vitamin D3 and its various metabolites to target organs and tissues. This is manifested by the ability of DBP to bind vitamin D3 and its metabolites with high affinity. In the present study we developed 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)ether (25-OH-D3-epoxide) as an affinity labeling reagent of human DBP (hDBP). Competitive radioligand binding assays of 25-OH-D3-epoxide with hDBP demonstrated that the binding affinity of this analog was similar to that of 25-hydroxyvitamin D3 (25-OH-D3). Incubation of 25-hydroxy[26(27)-3H]-vitamin D3-3 beta-(1,2-epoxypropyl)ether [[3H]25-OH-D3-epoxide] with hDBP covalently labeled the protein. When the incubation was carried out in the presence of a large excess of 25-OH-D3, labeling was removed completely. When human Cohn IV fraction, containing hDBP, was incubated with [3H]25-OH-D3-epoxide a single protein band, corresponding to hDBP, was labeled. Labeling was completely obliterated in the presence of a large amount of 25-OH-D3. However, an equivalent amount of 7-dehydrocholesterol had no effect on labeling. These results demonstrated that [3H]25-OH-D3-epoxide most probably labeled the vitamin D sterol-binding domain of hDBP.