Characterization of the bisphosphonate recognition site on hydroxyapatite using radioligand binding techniques with [14C]citric acid

Summary The present studies characterize the binding of [¹⁴C]citric acid to synthetic hydroxyapatite (HA) crystals. [¹⁴C]Citric acid specifically bound to HA and was dependent upon the concentration of HA in the assay. The binding of [¹⁴C]citric acid to HA reached equilibrium within 20 min and remained stable for at least 90 min. Dissociation of bound [¹⁴C]citric acid was biphasic in nature since both rapid and more slowly reversible binding components were detected. Saturation experiments also indicated that [¹⁴C]citric acid labeled two recognition sites with different affinity (Kd_H=42 nM and Kd_L=24,000 nM) and density (Bmax_H=161 fmol/g HA and Bmax_L=8.8 pmol/g HA). Ligand competition experiments revealed that compounds that are known to readily bind bone (e.g., sodium pyrophosphate, methylene diphosphonic acid, etidronate) potently inhibited the binding of [¹⁴C]citric acid to HA, whereas compounds known to have poorer affinity for bone (e.g., oxalic acid and GABA) did not. Computer analysis of these inhibition curves revealed specific ligand interactions at two different affinity recognition sites. The present results indicate that [¹⁴C]citric acid binds discrete sites on synthetic HA in a fashion consistent with a specific labeling of the bisphosphonate recognition site. Analysis of the binding of [¹⁴C]citric acid to HA provides a useful method to further explore the structure activity relationships of novel compounds that have binding affinity for bone.