Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
J Mol Biol. 2011 Sep 30;412(4):568-77. doi: 10.1016/j.jmb.2011.07.069. Epub 2011 Aug 4.
Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With β(2)-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins.
淀粉样纤维类似于晶体，通过成核和生长形成。由于成核的自由能势垒很高，淀粉样纤维的自发形成仅在长时间的滞后阶段后发生。超声处理可用于诱导淀粉样核形成，从而形成纤维，而使用带有硫黄素 T 荧光的微孔板读数器适合同时检测许多样品中的纤维。结合使用超声处理和微孔板读数器，我们提出了一种研究蛋白质形成淀粉样纤维潜力的有效方法。用β(2)-微球蛋白，一种导致透析相关淀粉样变性的淀粉样蛋白，在 pH 2.5 下几分钟内就形成了纤维。即使在中性 pH 条件下，也要经过 1.5 小时的滞后时间才形成纤维。结果表明，纤维形成是一种物理反应，主要受高自由能势垒的限制，超声处理可有效降低该势垒。这种方法将有助于开发一种用于蛋白质淀粉样变性的高通量检测方法。
