Noda Sayaka, So Masatomo, Adachi Masayuki, Kardos József, Akazawa-Ogawa Yoko, Hagihara Yoshihisa, Goto Yuji
Institute for Protein Research, Osaka University , Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Department of Biochemistry and MTA-ELTE NAP B Neuroimmunology Research Group, Eötvös Loránd University , Pázmány sétány 1/C, Budapest 1117, Hungary.
Biochemistry. 2016 Jul 19;55(28):3937-48. doi: 10.1021/acs.biochem.6b00231. Epub 2016 Jul 6.
Ultrasonication is considered one of the most effective agitations for inducing the spontaneous formation of amyloid fibrils. When we induced the ultrasonication-dependent fibrillation of β2-microglobulin and insulin monitored by amyloid-specific thioflavin T (ThT) fluorescence, both proteins showed a significant decrease in ThT fluorescence after the burst-phase increase. The decrease in ThT fluorescence was accelerated when the ultrasonic power was stronger, suggesting that this decrease was caused by the partial denaturation of preformed fibrils. The possible intermediates of denaturation retained amyloid-like morphologies, secondary structures, and seeding potentials. Similar denaturation intermediates were also observed when fibrils were denatured by guanidine hydrochloride or sodium dodecyl sulfate. The presence of these denaturation intermediates is consistent with the main-chain-dominated architecture of amyloid fibrils. Moreover, in the three types of denaturation experiments conducted, insulin fibrils were more stable than β2-microglobulin fibrils, suggesting that the relative stability of various fibrils is independent of the method of denaturation.
超声处理被认为是诱导淀粉样纤维自发形成的最有效搅拌方法之一。当我们通过淀粉样特异性硫黄素T（ThT）荧光监测诱导β2-微球蛋白和胰岛素的超声依赖性纤维化时，两种蛋白质在爆发期增加后ThT荧光均显著降低。当超声功率更强时，ThT荧光的降低加速，表明这种降低是由预先形成的纤维的部分变性引起的。变性的可能中间体保留了淀粉样形态、二级结构和种子形成潜力。当纤维被盐酸胍或十二烷基硫酸钠变性时，也观察到了类似的变性中间体。这些变性中间体的存在与淀粉样纤维的主链主导结构一致。此外，在进行的三种变性实验中，胰岛素纤维比β2-微球蛋白纤维更稳定，这表明各种纤维的相对稳定性与变性方法无关。
