Department of Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045, USA.
J Pharm Sci. 2011 Mar;100(3):836-48. doi: 10.1002/jps.22345. Epub 2010 Sep 22.
Solution conditions greatly affect the aggregation rate of a protein. Elucidating these influences provides insight into the critical factors governing aggregation. In this study, recombinant human botulinum protein antigen serotype C [rBoNTC (H(c))] was employed as a model protein. rBoNTC (H(c)) aggregated irreversibly during incubation at 42°C. The aggregation rate was studied as a function of solution conditions, including varying the pH from 3.5 to 8.0 and with or without 150 mM NaCl, 7.5% (w/v) trehalose, and 0.5 M urea. Some solution conditions retarded rBoNTC (H(c)) aggregation, whereas others accelerated aggregation, particularly acidic pH and addition of NaCl or urea. To better understand the mechanism by which these solution conditions influenced aggregation rates, the structure of rBoNTC (H(c)) was characterized using circular dichroism, fluorescence, and ultraviolet absorbance spectroscopies. Conformational stability was assessed from equilibrium urea-induced unfolding studies and by using differential scanning calorimetry (DSC). The activation energy of the aggregation reaction (E(a)) was estimated from an analysis of the heating-rate dependence of the thermal transition observed during DSC heating scans. Overall, for rBoNTC (H(c)), an inverse correlation was found between conformational stability and aggregation rate, as well as between the kinetic barrier to unfolding (i.e., E(a)) and aggregation rate.
溶液条件极大地影响蛋白质的聚集速率。阐明这些影响因素有助于深入了解控制聚集的关键因素。在本研究中，采用重组人肉毒梭菌蛋白抗原血清型 C [rBoNTC (H(c))]作为模型蛋白。rBoNTC (H(c))在 42°C 孵育时会发生不可逆聚集。研究了聚集速率作为溶液条件的函数，包括改变 pH 值从 3.5 到 8.0，以及有无 150 mM NaCl、7.5%（w/v）海藻糖和 0.5 M 脲。一些溶液条件会延缓 rBoNTC (H(c))的聚集，而另一些则会加速聚集，特别是酸性 pH 值和添加 NaCl 或脲。为了更好地理解这些溶液条件影响聚集速率的机制，使用圆二色性、荧光和紫外吸收光谱法对 rBoNTC (H(c))的结构进行了表征。通过平衡脲诱导的展开研究和差示扫描量热法（DSC）评估了构象稳定性。从 DSC 加热扫描过程中观察到的热转变的升温速率依赖性分析中估算了聚集反应的活化能 (E(a))。总的来说，对于 rBoNTC (H(c))，构象稳定性与聚集速率之间以及解折叠的动力学障碍（即 E(a))）与聚集速率之间呈反比关系。
