Inhibition of Calcium Carbonate and Phosphate Crystallization by Peptides Enriched in Aspartic Acid and Phosphoserine

Polyanionic peptide analogs of matrix proteins from biominerals were synthesized by automated, solid-phase methods. The peptides were used to evaluate the chemical requirements for inhibition of calcium carbonate and phosphate crystallization as measured using pH-drift and constant-composition assays. Continuous runs of negatively charged residues were required for maximum inhibitory activity. Asp15 was the optimum size for inhibition of CaCO3 crystal growth, but Asp(30 to 40) exhibited maximal inhibition of CaCO3 crystal nucleation. An hydrophobic domain of Alas added to Asp15 led to increased inhibition of CaCO3 crystal nucleation but had no effect on crystal growth. Adding the hydrophobic domain to Asp40 did not enhance inhibition of either CaCO3 nucleation or crystal growth. The results suggest that crystal nucleation can be suppressed through diffusion-limitation related to the presence of a barrier at the crystal/solution interface. Asp40 molecules seemed to fill both the crystal-binding sites and the zone of diffusion around them, with additional anionic or hydrophobic residues simply contributing excess mass without enhancing performance. CaCO3 crystal growth appeared not limited by diffusion but rather by some other process such as incorporation of already bound ions into the lattice. Phosphorylation of Nterminal serine residues by use of monochlorophosphoric acid produced H-PSer(1 to 3)-Asp20-OH, the most powerful inhibitor of both calcium carbonate and calcium phosphate formation yet discovered. This supports the importance of phosphorylated residues to the function of protein inhibitors of mineralization and suggests that the terminal residues may play a significant ole in the mechanism of inhibition.