不同pH对蛋清溶菌酶形成淀粉样纤维的影响

Effect of Different pH Conditions on Amyloid Fibrils Formation of Hen Egg-white Lysozyme

试验旨在研究不同pH对淀粉样纤维形成的影响。以蛋清溶菌酶(hen egg-white lysozyme,HEWL)为模型蛋白,在不同pH的甘氨酸(50 mmol/L)溶液中(57.0±0.1)℃孵育0~8 d,使其形成不同聚集程度的淀粉样纤维,pH设为2.0、6.5、7.5和8.0;用透射电镜定性观察淀粉样纤维生成状况,采用硫磺素T(ThT)荧光法和刚果红染色法测定淀粉样纤维生长状况,8-苯胺基-1-萘磺酸(ANS)荧光法测定淀粉样纤维疏水性变化,圆二色谱法测定二级结构转化,BeStSel软件预测β-折叠含量。结果显示,透射电镜下观察第8天纤维生长状况发现,pH为2.0时HEWL形成了大量的短杆状淀粉样纤维,同时ThT荧光法结果表明淀粉样纤维生长状况最好(P<0.01),且生长趋势呈时间依赖性;刚果红染色法显示,在490~510 nm出现红移、540 nm出现特征性肩峰,疏水性显著增强,并且二级结构转化明显,β-折叠含量从天然HEWL蛋白的6.1%增加到37.6%;pH 6.5组仅在透射电镜下观察到极少量的淀粉样纤维,pH 7.5组在视野中未观察到明显的淀粉样纤维,仅观察到大量的球状或杆状蛋白聚集体;pH 8.0组未产生淀粉样纤维。高温条件下,HEWL在强酸性(pH 2.0)条件较易形成淀粉样纤维,且此时疏水性和二级结构转化程度最高,这为解释朊病毒病及其他淀粉样变性疾病的淀粉样纤维形成机制提供了理论基础。

The aim of this study was to investigate the effect of pH conditions on amyloid fibrils formation.Hen egg-white lysozyme(HEWL)was used as the model protein in this study.HEWL was prepared in different pH conditions,such as pH 2.0,6.5,7.5 and 8.0.Then HEWL samples were incubated at(57.0±0.1)℃for 8 days to aggregate into amyloid fibrils.Morphology was imaged by transmission electron microscopy.Thioflavin T(ThT)and Congo red dye methods were used to detect the growth of HEWL aggregates,respectively.8-anilino-1-naphthalenesulfonic acid(ANS)fluorescence was used to quantify the hydrophobicity of formed HEWL fibrils.Circular dichroism was explored to measure the secondary structural transition and forecast theβ-sheet content.The results showed that HEWL at pH 2.0 solution produced large rod-shaped amyloid fibrils under transmission electron microscopy after incubating for 8 days,and ThT fluorescence results showed that the fibrils quantity at pH 2.0 were higher than other pH groups 2.0 groups(P<0.01),and the growth of amyloid fibrils was time-dependent.Furthermore,Congo red detected both the shift of absorption peak from 490 nm to 510 nm and characteristic shoulder peak at 540 nm,hydrophobicity andβ-sheet content were significantly increased,withβ-sheet content was increased from 6.1%of native HEWL to 37.6%of pH 2.0 HEWL.pH 6.5 group produced very fewer amyloid fibrils only under transmission electron microscopy.pH 7.5 group produced no visible amyloid fibrils,while visible abundant spherical or rod-shaped aggregated proteins appeared under transmission electron microscopy.However,pH 8.0 group produced no fibrils and aggregated proteins for any detected methods.The results suggested that HEWL easily aggregated into amyloid fibrils at pH 2.0 with the highest hydrophobicity and structural conversion,which provided basic data for the mechanisms of fibrillary aggregation of prion disease and other amyloidosis.