Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA.
J Pharm Sci. 2010 Feb;99(2):645-62. doi: 10.1002/jps.21869.
A global characterization of nonnative aggregation is presented for alpha-chymotrypsinogen A (aCgn) as a function of temperature (T), pH, and [NaCl]. Changes in unfolding free energy, native-state second osmotic virial coefficient (B(22)), and aggregation pathways and kinetics were qualitatively and quantitatively determined using a combination of size-exclusion chromatography, multi-angle laser light scattering, and circular dichroism and fluorescence spectroscopy. Results were analyzed quantitatively using multi-variate statistical models and a recently developed mechanistic model that naturally accounts for changes in aggregation pathway due to competition between unfolding, nucleation, chain polymerization, aggregate condensation, and phase separation. State diagrams are presented that show the natural progression between different aggregation behaviors or pathways. Together, the results show that pH and [NaCl] determine both the rates of aggregation and what aggregation behavior or pathway holds. In contrast, T affects primarily only aggregation rates, in large part due to changes in unfolding free energy. Finally, it is shown that B(22) correlates strongly with which type of aggregation pathway is followed, suggesting a potentially useful approach for predicting and controlling physical properties of the resulting aggregates.
本文对 alpha-糜蛋白酶原 A(aCgn) 的非天然聚集进行了全局特性分析，研究了温度 (T)、pH 和 [NaCl] 对其的影响。采用凝胶过滤色谱、多角度激光光散射和圆二色性和荧光光谱联用的方法，定性和定量地研究了变性自由能、天然状态第二渗透压系数 (B(22)) 以及聚集途径和动力学的变化。利用多元统计模型和最近开发的一种机制模型对结果进行了定量分析，该模型自然考虑了由于变性、成核、链聚合、聚集凝结和相分离之间的竞争而导致聚集途径发生变化的情况。本文还提出了状态图，显示了不同聚集行为或途径之间的自然进展。总之，结果表明 pH 和 [NaCl] 决定了聚集的速率和聚集行为或途径。相比之下，T 主要只影响聚集速率，这在很大程度上是由于变性自由能的变化。最后，结果表明 B(22)与所遵循的聚集途径类型密切相关，这表明这可能是一种预测和控制聚集产物物理性质的有用方法。
