Two proteins with gamma-carboxyglutamic acid in frog bone: isolation and comparative characterization

Two gamma-carboxyglutamic acid-containing proteins were purified from neutral (pH 7.5) EDTA-extract of frog, Rana catesbiana, cortical bone by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 chromatography and successive hydroxyapatite column chromatography. These two bone gamma-carboxyglutamic acid-containing proteins, termed osteocalcin, P-1 and P-2, had molecular weights of about 5100 and 4900, respectively, based on their amino-acid composition. Both species of osteocalcin have two gamma-carboxyglutamic acid residues, one disulfide bond, but there was no 4-hydroxyproline in either molecule. Each N-terminus of both proteins was acetylated and each C-terminal amino acid was lysine. The isoelectric points of P-1 and P-2 are 4.02 and 3.91, respectively, and their pI values shifted to more neutral pH in the presence of calcium ions. Equilibrium dialysis has indicated that each of these two proteins binds specifically 2 mol Ca2+, and nonspecifically more, 4-5 mol, Ca2+ in 0.02 M Tris-HCl/0.15 M NaCl (pH 7.4), at 4 degrees C. By the best-fitted calculation, P-1 had one high affinity Ca2+-binding site (Kd1 = 0.17 mM) and one lower affinity site (Kd2 = 0.29 mM), and P-2 contained one high affinity site (Kd1 = 0.154 mM) and one lower affinity site (Kd2 = 0.67 mM).