Incorporation of osteopontin peptide into kidney stone-related calcium oxalate monohydrate crystals: a quantitative study

Polyelectrolyte–crystal interactions regulate many aspects of biomineralization, including the shape, phase, and aggregation of crystals. Here, we quantitatively investigate the role of phosphorylation in interactions with calcium oxalate monohydrate crystals (COM), using synthetic peptides corresponding to the sequence 220–235 in osteopontin, a major inhibitor of kidney stone-related COM formation. COM formation is induced in the absence or presence of fluorescent-labeled peptides containing either no (P0), one (P1) or three (P3) phosphates and their adsorption to and incorporation into crystals determined using quantitative fluorimetry (also to determine maximum adsorption/incorporation), confocal/scanning electron microscopy and X-ray/Raman spectroscopy. Results demonstrate that higher phosphorylated peptides show stronger irreversible adsorption to COM crystals (P3: K⁰ ~ 66.4 × 10⁶ M⁻¹; P1: K⁰ ~ 29.4 × 10⁶ M⁻¹) and higher rates of peptide incorporation into crystals (maximum: P3: ~ 58.8 ng and P1: ~ 8.9 ng per µg of COM) than peptides containing less phosphate groups. However, crystals grown at that level of incorporable P3 show crystal-cleavage. Therefore, extrapolation of maximum incorporable P3 was carried out for crystals that are still intact, resulting in ~ 49.1 ng P3 µg⁻¹ COM (or ~ 4.70 wt%). Both processes, adsorption and incorporation, proceed via the crystal faces {100} > {121} > {010} (from strongest to weakest), with X-ray and Raman spectroscopy indicating no significant effect on the crystal structure. This suggests a process in which the peptide is surrounded by growing crystal matrix and then incorporated. In general, knowing the quantity of impurities in crystalline/ceramic matrices (e.g., kidney stones) provides more control over stress/strain or solubilities, and helps to categorize such composites.