Ca2+‐Binding Studies of the Phosphoprotein from Rat‐Incisor Dentine

Abstract

Rat incisor dentin was demineralized and extracted with 0.25 M EDTA containing protease inhibitors. The extract was purified by chromatography on DEAE-cellulose and sulfonated polystyrene. The Ca2+-binding properties of the phosphoprotein were studied by dynamic dialysis and by using a Ca2+-selective electrode. Two different binding sites were detected with Kd = 0.9 .times. 10-7 M and 1.1 .times. 10-5 M and displaying a Ca2+-binding capacity of 127 and 176 mol bound Ca2+/mol protein, respectively, assuming a MW of 30,000. Upon enzymatic dephosphorylation of the phosphoprotein, the highest affinity sites disappeared and those with the lowest affinity were reduced. The optimum for Ca2+ binding by the phosphoprotein occurred at pH 8.2. The specificity of the Ca2+ ion interaction with the phosphoprotein was investigated by studying the competitive nature of other divalent and monovalent cations. Ca2+ ions evidently were, to a large extent, displaced from the phosphoprotein by other cations in physiological concentrations.