The (ethylenedinitrilo)tetraacetic acid extracted phosphoprotein of bovine dentin contains 37.6 residue % of aspartic acid and 41.8 residue % serine, most of which is phosphoserine. The phosphoprotein binds calcium ions to both high- and low-affinity sites, with association constants 3.6 × 104 and 5.1 × 102 mol-1, respectively. Alkaline-hydrolysis studies showed that γ-carboxyglutamic acid was absent from this calcium-binding protein. The binding interactions are nonideal, showing a dependence of the number of high-affinity sites per milligram of protein on the protein concentration. This appears to be the result of a calcium-ion mediated concentration-dependent aggregation of the phosphoprotein. Conformational studies, using circular dichroism, show the protein to have a random chain conformation in aqueous solution. The addition of either methanol (to 50%, v/v) or Ca ion caused a change in the circular dichroism spectra, suggesting a transition to a more ordered extended chain conformation. 31P nuclear magnetic resonance spectra of the phosphoprotein showed resonances corresponding only to the presence of orthophosphates. Spectra determined during the titration of an alkaline solution of the sodium ion salt of the phosphoprotein showed the presence of a single set of acidic groups with a pKa of 6.8, also characteristic of orthophosphates. The addition of calcium ions caused a significant signal broadening of the orthophosphate resonance, indicating a specific calcium ion-orthophosphate interaction. A calcium-phosphoprotein complex precipitates upon standing at neutral pH at room temperature. Infrared spectra of this precipitate showed the presence of bands at 500-600 cm-1, characteristic of calcium-orthophosphate interaction. Examination of the infrared bands in the carboxylate region also established the presence of calcium-carboxylate interactions in the calcium-phosphoprotein complex. Both carboxylate and orthophosphate groups thus appear to be involved in providing high-affinity binding sites for calcium ions. In the trans-extended chain conformations suggested by the circular dichroism data, sheet-like arrays would present high-affinity calcium-binding faces that could initiate crystal phase growth.
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