Fourier-transform infrared spectroscopic study of Ca²⁺-binding to osteocalcin

Osteocalcin, the γ-carboxyglutamic acid-containing protein, which is the most abundant noncollagenous protein of bone and dentin, is considered to play roles in bone formation and remodeling. It is unclear how the γ-carboxyglutamic acid side-chains in osteocalcin coordinate to Ca²⁺, since the X-ray structure of osteocalcin is not available. Interactions between Ca²⁺ and the γ-carboxyglutamic acid side-chains in osteocalcin were investigated by Fourier-transform infrared spectroscopy. In the region of the antisymmetric stretches, the loss of intensity at 1574 cm^-1 and gain of intensity at 1600 cm^-1 were observed due to Ca²⁺-binding to osteocalcin. The spectral changes indicate that the γ-carboxyglutamic acid side-chains in osteocalcin coordinate to Ca²⁺ in the malonate chelation mode, where a Ca²⁺ interacts with two oxygen atoms, one from each of the two COO^- groups of a single γ-carboxyglutamic acid residue. Addition of Ca²⁺ does not cause any spectral change in the spectra of decarboxylated osteocalcin since the γ-carboxyglutamic acid residues are converted to the glutamic acid residues by chemical modification.