Use of surface plasmon resonance in the binding study of vitamin D, metabolites and analogues with vitamin D binding protein

Vitamin D-3 and its metabolites are lipophilic molecules with low aqueous solubility and must be transported bound to plasma carrier proteins, primarily to vitamin D binding protein (DBP). The biological functions of vitamin D-3 metabolites are intimately dependent on the protein, hence the importance of determining their affinity for DBP. In this study, we developed a novel procedure for measuring the kinetic and equilibrium constants of human-DBP with vitamin D-3 and three metabolites: 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3], 25-hydroxyvitamin D-3 (25OHD(3)) and 24,25-dihydroxyvitamin D-3 [24,25(OH)(2)D-3] by surface plasmon resonance (SPR). At the same time, five different analogues, synthetized in our laboratory, were evaluated in order to compare the affinity values with the metabolites. Real-time SPR measurements showed that 25OHD(3) and 24,25(OH)(2)D-3 had higher affinity (0.3 mu M) than 1,25(OH)(2)D-3 (5 mu M), with the higher affinity of 25OHD(3) and 24,25(OH)(2)D-3 due to dissociation constants 1 order of magnitude slower. In the case of the analogues, the affinity values were lower, with 1-hydroxy-25-nitro-vitamin D-3 (NO2-446), structurally closer to 1,25(OH)(2)D-3, showing the highest value with a K-D of 50 mu M. (24R)-1,25-dihydroxyvitamin-24-buthyl-28norvitamin D-2 (Bu-471) and (24R)-1,25-dihydroxyvitamin24- phenyl-28-norvitamin D-2 (Ph-491), structurally similar, had affinities of 180 and 170 mu M, respectively. (22R, 23E)-1hydroxy- 22-ethenyl-25-methoxy-23-dehydrovitamin D3 (MeO-455) and 1-h y d roxy-20(R)-[5 (S)-(2, 2 dimethyltetrahydropyran- 5-yl)]-22,23-dinor vitamin D-3 (Oxan-429) had affinities of 310 and 100 mu M, respectively. The binding of the metabolites and analogues was reversible allowing the rapid capture of data for replicates. The kinetic and equilibrium data for both the metabolites and the analogues fitted to the Langmuir model describing a 1:1 interaction.