The effect of proteins on hydroxyapatite dissolution

Studying proteins used to retard bone (or hydroxyapatite) dissolution could provide important design principles for the development of decalcification inhibitors and at the same time calcification promoters in orthopedics, cardiology, urology, and dentistry. The aim of this research is to study the effect of proteins known to have specific binding activity on hydroxyapatite (HA) dissolution. Statherin and osteopontin have been taken as reference proteins and synthetic HA as model substrate. Since HA hydrolysis produces protons in solution, a pH-Stat assay has been set up in order to follow and compare HA dissolution in the presence and the absence of model peptides. The effect of synthetic peptides corresponding to amino acid 220-235 and amino acid 65-80 of rat bone OPN have initially been considered. Due to the relevance of the presence of the phosphate group in calcium binding, OPN220-235 was synthesized in triphosphorylated (P3) and nonphosphorylated (P0) forms; OPN65-80 was synthesized in monophosphorylated (P-OPAR) and nonphosphorylated (OPAR) forms. Following the observation that highly acidic and phosphorylated peptides showed the most significant HA dissolution inhibition, also oligomers of asp and glu were prepared and tested. The substrate concentrations required to obtain 50% dissolution inhibition (IC50) were measured and compared: the most significant values was with P3, 6 [μg/ml], followed by P-OPAR,16 [μg/ml] , OPAR, 22 [μg/ml], P0 › 75 [μg/ml]. Poly -L-aspartic acid (MW=2,320), and poly -L-glutamic acid (MW=2,600), tested because of the significant affinity of polyanions to HA surface showed interesting inhibition properties. The corresponding (IC50) were: poly-Asp, 13 [μg/ml]; Poly-Glu, [18 μg/ml]. In an attempt to correlate the inhibition activity with the secondary structure of the peptides, structural informations were collected from their CD spectra. The secondary structure of these polyanions was investigated at 37ºC in P buffer at pH [5.25]. We found that poly-Asp was mostly disordered compared with the poly- Glu with the same length and contained roughly 20% poly-proline II helix. P3, has been shown to be one of the strong inhibitor of HA dissolution. In addition, the effects of statherin and DR9, which is 9-residue peptide derived from the N-terminal of the statherin have been studied. The IC50 measured were: statherin, 25 [μM]; DR9, 13 [μM]. DR9 was observed to have high affinity to HA surface compare to the statherin. In conclusion it has been shown that peptides structurally derived from natural salivary proteins and bone proteins are able to adsorb to hydroxyapatite surfaces and induce a decrease in the rate of dissolution of HA which strongly depends on their chemical structure. Since OPN and statherin possess relatively high proportions of aspartic acid, glutamic acid and phosphorylated serine residues one can assume that a combination of acidic residues and post-translational phosphorylation strongly influence the biomineral adsorption. The presence of the negative charges is imperative in the ability of acidic protein and peptides to adsorb to the HA surface and prevent the dissolution of HA.