Kotormán Márta, Simon Mária L, Borics Attila, Szabó Márton Richárd, Szabó Kitti, Szögi Titanilla, Fülöp Lívia
Department of Biochemistry and Molecular Biology, Faculty of Science and Informatics, University of Szeged, H-6726 Szeged, Középfasor 52, Hungary.
Protein Pept Lett. 2015;22(12):1104-10. doi: 10.2174/0929866522666151002154324.
The formation of amyloid-like fibrils was studied by using the well-known serine protease trypsin as a model protein in the presence of ethanol as organic solvent. Trypsin forms amyloid-like fibrils in aqueous ethanol at pH = 7.0. The dye Congo red (CR) was used to detect the presence of amyloid-like fibrils in the samples. The binding of CR to fibrils led to an increase in absorption intensity and a red shift in the absorption band of CR. Thioflavin T (ThT) and 8-anilino-1- naphthalenesulfonic acid (ANS) binding assays were employed to characterize amyloid-like fibril formation. The ThT binding assay revealed that the protein exhibited maximum aggregation in 60% (v/v) ethanol after incubation for 24 h at 24 (o)C. The ANS binding results indicated that the hydrophobic residues were more exposed to the solvent in the aggregated form of the protein. The effects of polyethylene glycol (PEG) on the formation of amyloid-like fibrils was studied in vitro. The aggregation of trypsin was followed via the kinetics of aggregation, the far-UV circular dichroism (CD) and transmission electron microscopy (TEM) in the presence and absence of PEG. The CD measurements indicated that the protein aggregates have a cross-beta structure in 60% ethanol. TEM revealed that trypsin forms fibrils with a thread-like structure. The inhibitory effect of PEG on the aggregation of trypsin increased with rising PEG concentration. PEG therefore inhibits the formation of amyloid-like fibrils of trypsin in aqueous ethanol.
以著名的丝氨酸蛋白酶胰蛋白酶作为模型蛋白，在乙醇作为有机溶剂存在的情况下，研究了淀粉样纤维的形成。胰蛋白酶在pH = 7.0的乙醇水溶液中形成淀粉样纤维。使用刚果红（CR）染料检测样品中淀粉样纤维的存在。CR与纤维的结合导致吸收强度增加和CR吸收带发生红移。采用硫黄素T（ThT）和8-苯胺基-1-萘磺酸（ANS）结合试验来表征淀粉样纤维的形成。ThT结合试验表明，该蛋白在24℃孵育24小时后，在60%（v/v）乙醇中表现出最大聚集。ANS结合结果表明，在蛋白的聚集形式中，疏水残基更暴露于溶剂中。在体外研究了聚乙二醇（PEG）对淀粉样纤维形成的影响。在有和没有PEG的情况下，通过聚集动力学、远紫外圆二色性（CD）和透射电子显微镜（TEM）跟踪胰蛋白酶的聚集情况。CD测量表明，蛋白聚集体在60%乙醇中具有交叉β结构。TEM显示胰蛋白酶形成具有线状结构的纤维。PEG对胰蛋白酶聚集的抑制作用随着PEG浓度的升高而增加。因此，PEG抑制了胰蛋白酶在乙醇水溶液中淀粉样纤维的形成。
