Amani Samreen, Naeem Aabgeena
Department of Biochemistry, Faculty of Life Science, Aligarh Muslim University, Aligarh, 202002, India,
Amino Acids. 2014 Aug;46(8):1839-51. doi: 10.1007/s00726-014-1698-y. Epub 2014 Apr 12.
Aggregation of protein into insoluble intracellular complexes and inclusion bodies underlies the pathogenesis of human neurodegenerative diseases. Importance of cytochrome c (cyt c) arises from its involvement in apoptosis, sequence homology and for studying molecular evolution. A systemic investigation of polyethylene glycol (PEG) and trifluoroethanol (TFE) on the conformational stability of cyt c as a model hemeprotein was made using multi-methodological approach. Cyt c exists as molten globule (MG) at 60% PEG-400 and 40% TFE as confirmed by far-UV CD, attenuated total reflection Fourier transform infrared spectroscopy, Trp environment, 8-anilino-1-naphthalene-sulfonic acid (ANS) binding and blue shift in the soret band. Q-band splitting in MG states specifies conformational changes in the hydrophobic heme-binding pocket. Aggregates were detected at 90% PEG-400 and 50% TFE as confirmed by increase thioflavin T and ANS fluorescence and shift in Congo red absorbance. Detection of prefibrils and protofibrils at 90% PEG-400 and 50% TFE was possible after 72-h incubation. Single cell gel electrophoresis of prefibrils and protofibrils showed DNA damage confirming their toxicity and potential health hazards. Scanning electron microscopy and XRD analysis confirmed prefibrillar oligomers and protofibrils of cyt c.
蛋白质聚集成不溶性细胞内复合物和包涵体是人类神经退行性疾病发病机制的基础。细胞色素c（cyt c）的重要性源于其参与细胞凋亡、序列同源性以及用于研究分子进化。采用多方法对聚乙二醇（PEG）和三氟乙醇（TFE）对作为模型血红素蛋白的cyt c构象稳定性的影响进行了系统研究。通过远紫外圆二色光谱、衰减全反射傅里叶变换红外光谱、色氨酸环境、8-苯胺基-1-萘磺酸（ANS）结合以及Soret带蓝移证实，在60% PEG - 400和40% TFE条件下，cyt c以熔球态（MG）存在。MG状态下的Q带分裂表明疏水血红素结合口袋发生了构象变化。通过硫黄素T和ANS荧光增强以及刚果红吸光度的变化证实，在90% PEG - 400和50% TFE条件下检测到了聚集体。在90% PEG - 400和50% TFE条件下孵育72小时后，可以检测到前纤维和原纤维。前纤维和原纤维的单细胞凝胶电泳显示DNA损伤，证实了它们的毒性和潜在健康危害。扫描电子显微镜和XRD分析证实了cyt c的前纤维状寡聚体和原纤维。
