The effect of Pb2+ on the structure and hydroxyapatite binding properties of osteocalcin

Lead toxicity is a major environmental health problem in the United States. Bone is the major reservoir for body lead. Although lead has been shown to impair bone metabolism in animals and at the cellular level, the effect of Pb²⁺ at the molecular level is largely unknown. We have used circular dichroism (CD), and a hydroxyapatite binding assay to investigate the effect of Pb²⁺ on the structure and mineral binding properties of osteocalcin, a noncollagenous bone protein. The CD data indicate Pb²⁺ induces a similar structure in osteocalcin as Ca²⁺ but at 2 orders of magnitude lower concentration. These results were explained by the more than 4 orders of magnitude tighter binding of Pb²⁺ to osteocalcin (K_d=0.085 μM) than Ca²⁺ (K_d=1.25 mM). The hydroxyapatite binding assays show that Pb²⁺ causes an increased adsorption to hydroxyapatite, similar to Ca²⁺, but at 2–3 orders of magnitude lower concentration. Low Pb²⁺ levels (1 μM) in addition to physiological Ca²⁺ levels (1 mM) caused a significant (40%) increase in the amount of mineral bound osteocalcin as compared to 1 mM Ca²⁺ alone. These results suggest a molecular mechanism of Pb²⁺ toxicity where low Pb²⁺ levels can inappropriately perturb Ca²⁺ regulated processes. In-vivo, the increased mineral bound osteocalcin could play a role in the observed low bone formation rates and decreased bone density observed in Pb²⁺-intoxicated animals.