Department of Physics and Astronomy, University of Western Ontario, London, Ontario, Canada.
PLoS One. 2013;8(2):e56764. doi: 10.1371/journal.pone.0056764. Epub 2013 Feb 14.
We study the effect of isoforms of osteopontin (OPN) on the nucleation and growth of crystals from a supersaturated solution of calcium and phosphate ions. Dynamic light scattering is used to monitor the size of the precipitating particles and to provide information about their concentration. At the ion concentrations studied, immediate precipitation was observed in control experiments with no osteopontin in the solution, and the size of the precipitating particles increased steadily with time. The precipitate was identified as hydroxyapatite by X-ray diffraction. Addition of native osteopontin (nOPN) extracted from rat bone caused a delay in the onset of precipitation and reduced the number of particles that formed, but the few particles that did form grew to a larger size than in the absence of the protein. Recombinant osteopontin (rOPN), which lacks phosphorylation, caused no delay in initial calcium phosphate precipitation but severely slowed crystal growth, suggesting that rOPN inhibits growth but not nucleation. rOPN treated with protein kinase CK2 to phosphorylate the molecule (p-rOPN) produced an effect similar to that of nOPN, but at higher protein concentrations and to a lesser extent. These results suggest that phosphorylations are critical to OPN's ability to inhibit nucleation, whereas the growth of the hydroxyapatite crystals is effectively controlled by the highly acidic OPN polypeptide. This work also demonstrates that dynamic light scattering can be a powerful tool for delineating the mechanism of protein modulation of mineral formation.
我们研究了骨桥蛋白（OPN）异构体对钙和磷酸盐离子过饱和溶液中晶体成核和生长的影响。动态光散射用于监测沉淀颗粒的大小，并提供有关其浓度的信息。在研究的离子浓度下，在没有骨桥蛋白的溶液中进行对照实验时，立即观察到沉淀，沉淀颗粒的大小随时间稳步增加。通过 X 射线衍射，将沉淀鉴定为羟基磷灰石。添加从大鼠骨骼中提取的天然骨桥蛋白（nOPN）会延迟沉淀的开始，并减少形成的颗粒数量，但形成的少数颗粒会生长到比没有蛋白质时更大的尺寸。缺乏磷酸化的重组骨桥蛋白（rOPN）不会延迟初始磷酸钙沉淀，但会严重减缓晶体生长，表明 rOPN 抑制生长而不抑制成核。用蛋白激酶 CK2 处理 rOPN 使其磷酸化（p-rOPN）产生类似于 nOPN 的效果，但在更高的蛋白质浓度下效果较小。这些结果表明，磷酸化对于 OPN 抑制成核的能力至关重要，而羟基磷灰石晶体的生长则受到高度酸性的 OPN 多肽的有效控制。这项工作还表明，动态光散射可以成为描绘蛋白质调节矿物质形成机制的有力工具。
