Date of Award


Degree Type


Degree Name

Master of Science




Dr. Graeme Hunter

Second Advisor

Dr. Harvey Goldberg


Biominerals often possess features such as size, shape and crystallinity that are distinct from their abiotically formed equivalents. Organisms are known to achieve this by regulating the crystal growth process. To understand the specific structural features of the phosphoprotein osteopontin (OPN) responsible for its inhibition of hydroxyapatite (HAP) growth, we used a constant-composition seeded-growth assay. In addition to testing the HAP growth-inhibiting effects of the highly phosphorylated bovine milk OPN and the nonphosphorylated rat recombinant OPN, we have also tested the effects of synthetic peptides corresponding to amino acids 220-235 (pSHEpSTEQSDAIDpSAEK) and amino acids 65-80 (pSHDHMDDDDDDDDDGD) of rat bone OPN. OPN220-235 was synthesized in triphosphorylated (P3) and nonphosphorylated (P0) forms; OPN65-80 was synthesized in monophosphorylated (P-OPAR) and nonphosphorylated (OPAR) forms. Also studied was poly-L-aspartic acid (poly-Asp) of 11 kDa. Highly acidic and phosphorylated peptides/proteins were observed to be the most potent inhibitors of HAP growth. The 50% inhibitory concentrations measured were: poly-Asp, 1.45 pg/ml; P- OPAR, 1.65 pg/ml; milk OPN, 1.94 pg/ml; P3, 2.03 pg/ml; OPAR, 2.47 pg/ml; recombinant OPN, 5.23 pg/ml; and P0, >75 pg/ml. The thermodynamics of OPN and OPN-sequence-derived peptides adsorbing onto the surface of HAP was probed using isothermal titration calorimetry. At 37°C, adsorption of all strong inhibitors is characterized by an endothermic enthalpy that rapidly diminishes to zero when HAP surface coverage approached 10%. The free energy o f adsorption is dominated by large positive entropy change, likely resulting from the liberation of water molecules from the HAP surface. It appears that negative charge, whether contributed by phosphorylated or acidic residues, is a prerequisite for the entropically driven adsorption to and growth

inhibition of HAP.



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