基于ThT荧光寿命检测蛋清溶菌酶蛋白寡聚体
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摘要
采用时间分辨荧光技术,检测了不同形态蛋白聚集体的荧光染料硫磺素T(ThT)荧光寿命.利用蛋清溶菌酶体外制备了蛋白聚集体;采用透射电子显微镜(TEM)及ThT稳态荧光检测了结合蛋白纤维生长的动力学曲线,确定其形成寡聚体及纤维样聚集体的特征和时间.通过时间相关单光子计数(TCSPC)技术测定了蛋清溶菌酶单体、寡聚体和淀粉样纤维的ThT荧光寿命曲线,并拟合、计算其荧光寿命.根据圆二色谱(CD)分析结果推测聚集体的结构不同导致其与ThT的结合状态不同,从而影响ThT荧光寿命.结果表明,通过测定ThT荧光寿命可以区分蛋白单体、寡聚体和纤维样聚集体,并监测蛋白寡聚体的形成,为后续病理蛋白聚集过程中形成寡聚体物质的监测提供了研究基础.
Thioflavin T( ThT) fluorescence lifetime was used to detect aggregates states by time-resolved fluorescence. The protein aggregation was made by the egg white lysozyme in vitro. The characteristics of the oligomer and fibril were determined by means of transmission electron microscopy,ThT steady-state fluorescence and the growth kinetic curve of aggregation. The time-correlated single-photon counting technique was used to measure the fluorescence lifetime of ThT incubation with the aggregations. It calculated the fluorescence lifetime by fitting to the double exponential equation. The results of circular dichroism speculated that the structure of different aggregates was different and affected the fluorescence lifetime of ThT. It demonstrated that ThT fluorescence lifetime can distinguish protein monomers,oligomers and fibrils,and monitor the formation of protein oligomers. The results provide a basis for the monitoring of oligomer substances in the proess of sub-sequent pathological protein aggregation.
Thioflavin T( ThT) fluorescence lifetime was used to detect aggregates states by time-resolved fluorescence. The protein aggregation was made by the egg white lysozyme in vitro. The characteristics of the oligomer and fibril were determined by means of transmission electron microscopy,ThT steady-state fluorescence and the growth kinetic curve of aggregation. The time-correlated single-photon counting technique was used to measure the fluorescence lifetime of ThT incubation with the aggregations. It calculated the fluorescence lifetime by fitting to the double exponential equation. The results of circular dichroism speculated that the structure of different aggregates was different and affected the fluorescence lifetime of ThT. It demonstrated that ThT fluorescence lifetime can distinguish protein monomers,oligomers and fibrils,and monitor the formation of protein oligomers. The results provide a basis for the monitoring of oligomer substances in the proess of sub-sequent pathological protein aggregation.
引文
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[2] Pinney J. H.,Hawkins P. N.,Ann. Clin. Biochem.,2012,49(3),229—241
[3] Stefani M.,FEBS J.,2010,277(22),4602—4613
[4] Song S. M.,Ma X. W.,Zhou Y. H.,Xu M. T.,Shuang S. M.,Dong C.,Chem. Res. Chinese Universities,2016,32(2),172—177
[5] Gharibyan A. L.,Zamotin V.,Yanamandra K.,Moskaleva O. S.,Margulis B. A.,Kostanyan I. A.,Morozova-Roche L. A.,Mol. Biol. J.,2007,365,1337—1349
[6] Sarroukh R.,Goormaghtigh E.,Ruysschaert J. M.,Raussens V.,Biochim. Biophys. Acta,2013,1828,2328—2338
[7] Biancalana M.,Makabe K.,Koide A.,Molecular S.,Journal of Molecular Biology,2009,385(4),1052—1063
[8] Verma M.,Vats A.,Taneja V.,Annals of the Indian Academy of Neurology,2015,18,138—145
[9] Akihito H.,Hiroyuki A.,Akio K.,Biosci. Biotechol. Biochem.,2008,72(6),1523—1530
[10] Frare E.,Mossuto M. F.,de Laureto P. P.,Tolin S.,Menzer L.,Dumoulin M.,Dobson C. M.,Fontana A.,Mol. Biol. J.,2009,387,17—27
[11] Orte A.,Birkett N. R.,Clarke R. W.,Devlin G. L.,Dobson C. M.,Klenerman D.,Proc. Natl. Acad Sci. USA,2008,105,14424—14429
[12] Landau M.,Sawaya M. R.,Faull K. F.,Laganowsky A.,Jiang L.,Sievers S. A.,Liu J.,Barrio J. R.,Eisenberg D.,PLoS Biol.,2011,9(6),e1001080
[13] Song S.,Wang Y.,Xiong L.,Qu L.,Xu M.,Chem. Res. Chinese Universities,2013,29(1),20—25
[14] David J. L.,Moa S. W.,Melina G. G.,Fredrik W.,Elin K. E.,Biochem. Biophys. Res. Commun.,2015,458,418—423
[15] Hanczyc P.,Sznitko L.,Zhong C.,Heeger A.,ACS Photonics. J.,2015,2,1755—1762
[16] Dumoulin M.,Kumita J. R.,Dobson C. M.,Acc. Chem. Res.,2006,39,603—610
[17] Biancalana M.,Koide S.,Biochim. Biophys. Acta,2010,1804,1405—1412
[18] Amdursky N.,Erez Y.,Huppert D.,Acc. Chem. Res.,2012,45(9),1548—1557
[19] Singh P. K.,Mora A. K.,Nath S.,Chem. Commun.,2015,51,14042—14045
[20] Xue W. F.,Hellewell A. L.,Gosal W. S.,Biol. Chem. J.,2009,284,34272—34282
[21] Chen Y. C.,Pu Y. C.,Hsu Y. J.,J. Phys. Chem. C,2012,116,2967—2975
[22] Sznitko L.,Hanczyc P.,Mysliwiec J.,Samoc M.,Appl. Phys. Lett.,2015,106,023702