In vitro mineral induction by immobilized phosphoprotein : Effect of phosphate group on interfacial tension for mineral induction

Phosphoproteins are thought to play a primary role in the deposition of mineral on the collagen of dentin. We previously reported that immobilized phosphoproteins reduced activation energy for mineral induction in metastable mineralizing solutions, and that phosphate ester was essential for mineral induction. Interfacial tension is a good measure to evalute the mineral induction potential because it is a function of the activation energy. The purpose of this study was to determine the effect of phosphate ester on interfacial tension for mineral nucleation by immobilized phosphoprotein. Phosvitin from egg yolk was used as a model phophoprotein in this study. Phosvitin was cross-linked to agarose beads with divinyl sulfone. A portion of the cross-linked phosvitin was partially dephosphorylated with potato acid phosphatase. Then, samples were incubated at 37℃ in metastable solutions that do not spontaneously precipitate, and the mineral induction time was determined in the samples. The mineral formed was confirmed by X-ray diffraction to be hydroxyapatite. Using classical nucleation theory, the interfacial tension for hydroxyapatite nucleation by intact phosvitin was determined to be 91.3ergs/cm^2, it was 93.2 ergs/cm^2 for 50%-dephosphorylated phosvitin, and 98.1 ergs/cm^2 for 70%-dephosphorylated phosvitin. Mineral formation was not induced by phosvitin that had been 94%-dephosphorylated. These results indicate that the potential of hydroxyapatite nucleation of phosvitin is high as long as it has minimum number of phosphate esters for hydroxyapatite nucleation, and that the interfacial tension for hydroxyapatite nucleation by immobilized phosphoprotein is dependent on the degree of phosphorylation