Katayama Derrick S, Nayar Rajiv, Chou Danny K, Valente Joseph J, Cooper Julianne, Henry Charles S, Vander Velde David G, Villarete Lorelie, Liu C P, Manning Mark Cornell
Center for Pharmaceutical Biotechnology, University of Colorado Health Sciences Center, Denver, CO, USA.
J Pharm Sci. 2006 Jun;95(6):1212-26. doi: 10.1002/jps.20471.
It is now becoming apparent that a common pathway of protein aggregation involves the unimolecular structural rearrangement from the native state to a slightly expanded aggregation-competent species. It is the goal of this study to understand the aggregation and the effects of buffer on the stability of IFN-tau. In this study, the thermally-induced aggregation of interferon-tau (IFN-tau) is described. By monitoring the aggregation rate in the presence of increasing amounts of sucrose, the relative change in surface area (Deltas) for conversion to the aggregation-competent state can be determined. Under conditions of pH 7 and in 20 mM buffer, the protein displays different aggregation rates depending on the nature of the buffer species. The protein aggregates mostly quickly in phosphate buffer, slower in the presence of Tris and slowest in the presence of histidine. The largest value for Deltas occurs for the histidine-containing samples, where aggregation proceeds via a slightly expanded aggregation competent state with a surface area increase of 7.6%. Furthermore, it appears that histidine binds to the native state of IFN-tau, thereby stabilizing the native state and retarding aggregation. Measurement of the second virial coefficient, B(22), for different formulations indicates that inclusion of histidine has only a small effect on repulsion between protein molecules, suggesting that colloidal stabilization is not the dominant mechanism for stabilization of IFN-tau. This study represents the first detailed biophysical study of specific buffer-induced stabilization, resulting in shifting the equilibrium towards the native state and away form the expanded aggregation-competent species.
现在越来越明显的是，蛋白质聚集的一个共同途径涉及从天然状态到稍微扩展的具有聚集能力的物种的单分子结构重排。本研究的目的是了解聚集情况以及缓冲液对IFN-tau稳定性的影响。在本研究中，描述了热诱导的干扰素-tau（IFN-tau）聚集。通过监测在蔗糖量增加的情况下的聚集速率，可以确定转化为具有聚集能力状态时表面积的相对变化（ΔS）。在pH 7和20 mM缓冲液的条件下，蛋白质根据缓冲液种类的性质显示出不同的聚集速率。蛋白质在磷酸盐缓冲液中聚集最快，在Tris存在下较慢，在组氨酸存在下最慢。含组氨酸的样品的ΔS值最大，在该样品中聚集通过表面积增加7.6%的稍微扩展的具有聚集能力的状态进行。此外，似乎组氨酸与IFN-tau的天然状态结合，从而稳定天然状态并延缓聚集。对不同配方的第二维里系数B(22)的测量表明，加入组氨酸对蛋白质分子之间的排斥作用影响很小，这表明胶体稳定不是IFN-tau稳定的主要机制。这项研究代表了对特定缓冲液诱导的稳定化的首次详细生物物理研究，导致平衡向天然状态移动并远离扩展的具有聚集能力的物种。
