Amelogenins are the major secreted protein components of the mineralizing enamel extracellular matrix, and are critical for enamel crystal formation. Fluorosed enamel is more porous, possibly related to altered amelogenin modulated crystal growth. Objective: To study the effect of fluoride in the interaction between full-length amelogenin and apatite crystals. Methods: Hydroxyapatite (HAP), 3% carbonated hydroxyapatite (CAP) and fluoride-containing 3% carbonated hydroxyapatite (F-CAP) were synthesized, and characterized by specific surface area measurement, fourier transform-infrared reflection (FT-IR) spectroscopy and fluoride assay. Recombinant human amelogenin (rh174) was overexpressed in E. coli, purified, and characterized by SDS-PAGE, Western blot and mass spectrometry. The synthetic apatites and rh174 were added into 20 mM Tris-HCl, pH 7.5 for 1 h at 25 °C, then centrifuged at 4000 rpm for 5 min. The rh174 that bound to apatite was visualized by SDS-PAGE and quantified by the Bradford method. Direct binding of fluoride to rh174 was measured by isothermal titration calorimetry (ITC) analysis. Statistical analysis (ANOVA) was conducted for triplicates per reaction. Results: No significant difference was found in the binding of rh174 to HAP and CAP. However, F-CAP bound significantly more rh174 than CAP (P<0.01). Addition of NaF (1000 and 2000 ppm F^-) into the buffer significantly decreased bindings of rh174 to both CAP and F-CAP as compared to buffer without added F (P<0.01). ITC calorimetry showed no direct interaction between rh174 and fluoride ions. Conclusion: We suggested that amelogenin binds to calcium ions in the apatite lattice. Fluoride incorporation in the apatite lattice results in a more compact crystal structure and enhances amelogenin binding.

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