Department of Pharmaceutical Sciences and Center for Pharmaceutical Biotechnology, School of Pharmacy, University of Colorado Denver, 12700 E 19th Ave., C238-P15, Aurora, Colorado 80045, USA.
J Pharm Sci. 2011 May;100(5):1679-89. doi: 10.1002/jps.22426. Epub 2011 Jan 12.
Multidose protein formulations require an effective antimicrobial preservative (AP) to inhibit microbial growth during long-term storage of unused formulations. m-cresol (CR) is one such AP, but it has been shown to cause protein aggregation. However, the fundamental physical mechanisms underlying such AP-induced protein aggregation are not understood. In this study, we used a model protein cytochrome c to identify the protein unfolding that triggers protein aggregation. CR induced cytochrome c aggregation at preservative concentrations that are commonly used to inhibit microbial growth. Addition of CR decreased the temperature at which the protein aggregated and increased the aggregation rate. However, CR did not perturb the tertiary or secondary structure of cytochrome c. Instead, it populated an "invisible" partially unfolded intermediate where a local protein region around the methionine residue at position 80 was unfolded. Stabilizing the Met80 region drastically decreased the protein aggregation, which conclusively shows that this local protein region acts as an aggregation "hotspot." On the basis of these results, we propose that APs induce protein aggregation by partial rather than global unfolding. Because of the availability of site-specific probes to monitor different levels of protein unfolding, cytochrome c provided a unique advantage in characterizing the partial protein unfolding that triggers protein aggregation.
多剂量蛋白质制剂需要有效的抗菌防腐剂 (AP) 来抑制未使用制剂在长期储存过程中的微生物生长。间甲酚 (CR) 就是这样一种 AP，但它已被证明会导致蛋白质聚集。然而，这种 AP 诱导蛋白质聚集的基本物理机制尚不清楚。在这项研究中，我们使用模型蛋白细胞色素 c 来确定引发蛋白质聚集的蛋白质展开。在通常用于抑制微生物生长的防腐剂浓度下，CR 诱导细胞色素 c 聚集。添加 CR 降低了蛋白质聚集的温度并增加了聚集速率。但是，CR 并没有扰乱细胞色素 c 的三级或二级结构。相反，它形成了一种“不可见”的部分展开的中间状态，其中第 80 位甲硫氨酸残基周围的局部蛋白质区域展开。稳定 Met80 区域可大大降低蛋白质聚集，这明确表明该局部蛋白质区域是聚集的“热点”。基于这些结果，我们提出 AP 通过部分而不是整体展开来诱导蛋白质聚集。由于有可用的位点特异性探针来监测不同水平的蛋白质展开，细胞色素 c 在表征引发蛋白质聚集的部分蛋白质展开方面具有独特的优势。
