Molecular Mechanism for the Formation of Protein Aggregation Studied by High Pressure Infrared and Raman Spectroscopes

用傅立叶变换红外光谱法从二级结构的角度研究了压力和热诱导的马血清白蛋白（ESA）聚集体。我们显示了热和压力诱导的ESA聚集体之间的二级结构差异。ESA的热诱导不可逆聚集体由分子间β-折叠结构组成，不与硫磺素T和刚果红结合而形成无定形聚集体。另一方面，压力诱导的可逆聚集体由无规结构组成，也是无定形形式。通过比较二硫键在天然和还原条件下对ESA的压力效应，我们证明了二硫键对结构柔性的限制是压力诱导聚集可逆性的一个重要因素。一是压力对60℃孵育不同时间形成的胰岛素淀粉样蛋白（早期或成熟淀粉样蛋白原纤维）二级结构变化的影响;二是压力对胰岛素淀粉样蛋白原纤维形成速率的影响。明确了早期孵育的淀粉样蛋白原纤维对压力不稳定，但粪肥淀粉样蛋白原纤维对压力相当稳定，即使在高于1GPa的压力下。淀粉样纤维形成过程中的活化体积为较大的正值。

Pressure-and heat-induced aggregates of equine serum albumin (ESA) have been studied from the secondary stractural viewpoints using FT-IR spectroscopy. We showed the secondary structural differences between heat-and pressure0induced aggregates of ESA. The heat-induced irreversible aggregates of ESA are composed of the intermolecular β-sheet structure without binding thioflavin T and Congo red to be amorphous form. On the other hand, the pressure-induced reversible aggregates are composed of the random structure to be also amorphous form. From the comparison of pressure effects on ESA in native and reducing conditions of disulfide bridges, we demonstrate that the restriction of structure flexibility by disulfide bridges is an important factor for the reversibility of the pressure-induced aggregationThere were also two pressure effects on the secondary structure of insulin amyloid fibrils using FT-IR spectroscopy. One is the pressure effect on the secondary structural changes of insulin amyloid formed at different incubation time at 60℃ (early or mature amyloid fibrils) Another is the pressure effects on the formational rate of insulin amyloid fibrils. It was clarified that early incubated amyloid fibril was unstable against pressure but manure amyloid fibril quite stable against pressure, even under above 1 GPa. And the activation volume on the process of amyloid fibril formation was large positive values.