卵清溶菌酶淀粉样纤维形成的研究

Study of hen egg-white lysozyme fibrillation

淀粉样纤维与老年性痴呆症、帕金森病和非神经性组织淀粉样变性病等人类疾病相关。运用ThT荧光、刚果红结合、远紫外圆二色、透射电镜的方法研究了不同条件下卵清溶菌酶（HEWL）淀粉样纤维的形成。实验结果表明pH值2．0是HEWL淀粉样纤维形成的必要条件，HEWL淀粉样纤维的形成是一个典型的浓度依赖型过程。三氟乙醇（TFE）对HEWL淀粉样纤维形成影响结果表明中低浓度（低于40％）的TFE加速了溶菌酶淀粉样纤维的形成，其中5％～15％（v／v）的TFE促进效果最为显著，大大缩短了淀粉样纤维的成核期；而高浓度的TFE（50％）则完全抑制了溶菌酶淀粉样纤维的形成。透射电镜直接观察了溶菌酶淀粉样纤维的形态，不加TFE时溶菌酶淀粉样纤维聚集成簇，形成相互缠绕的成熟纤维，而10％的TFE存在时，观察到的形态则主要是短的原纤维，且没有发生纤维的相互交联实验。结果表明溶菌酶形成相互缠绕的成熟纤维主要由弱的疏水相互作用来驱动。

Amyloid fibrils are associated with Alzheimer＇s disease, Parkinson＇s disease, and nonneuropathic systemic amyloidosis. The amyloid formation of hen egg-white lysozyme（HEWL） has been examined by thioflavin T binding, Congo red binding, far-UV circular dichroism （CD） and transmission electron microscopy. The results showed that pH 2.0 was necessary for the HEWL fibrillation, and the process of HEWL amyloid formation was concentration-dependant. The data about effect of trifluoroethanol on the HEWL fibrillation indicated that TFE at low and moderate concentrations （ smaller than 40% ） accelerated amyloid formation of lysozyme, and 5% - 15% （v/v） TFE shortened the stage of nucleation phase of amyloid formation most remarkably. However, amyloid formation of lysozyme was completely impeded by TFE at high concentration （50%）. Transmission electron microscopy was employed to study the morphology of HEWL samples incubated under different conditions. When lysozyme was incubated in the absence of TFE, twisted amyloid fibrils of lysozyme in bundles were observed. In contrast, when lysozyme was incubated in 10% TFE, thin and long mature fibrils and some granular aggregates were observed. The experimental results showed that the formation of twisted mature fibrils was driven predominantly by weak hydrophobic interactions.