Conformational changes of bovine bone osteonectin induced by interaction with calcium

Abstract

To clarify calcium-induced conformational changes in bovine bone osteonectin, the protein was labeled with fluorescein isothiocyanate (FITC) in the presence and absence of calcium. By calcium titration using fluorescence spectrometry, it was demonstrated that FITC-osteonectin labeled in the presence of 2 mM CaCl2 showed a much higher affinity for calcium ions than did that labeled in the absence of calcium ions. The midpoint for completion of the increase in the intrinsic fluorescence (K0.5) of the two were 1 x 10(-7) M and 5 x 10(-7) M, respectively. By tryptic digestion and isolation of the fluorescent peptide of both FITC-osteonectins, the site of FITC-labeling was determined to be Lys174. Furthermore, it was found that the efficacy of labeling in this specific binding site was three times higher in the FITC-osteonectin labeled in the presence of 2 mM CaCl2 than in that labeled in the absence of calcium. The results indicate that in the presence of 2 mM CaCl2 the microenvironment around Lys174 of osteonectin was more open to modification than in the absence of calcium.