Tyrosine Autofluorescence as a Measure of Bovine Insulin Fibrillation
Tyrosine Autofluorescence as a Measure of Bovine Insulin Fibrillation
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DOI：
10.1016/j.bpj.2009.07.064
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发表时间：
2009-11-04
影响因子：
3.4
通讯作者：
Dunstan, Dave E.
中科院分区：
文献类型：
--
作者：
Bekard, Innocent B.;Dunstan, Dave E.
研究胰岛素和蛋白质的部分解折叠和原纤化的传统方法分别涉及使用染料1-苯胺基萘-8-磺酸（ANS）和硫磺素T（ThT）。我们比较了胰岛素原纤化过程中ThT、ANS、光散射和固有Tyr荧光的动力学特征。数据显示，伴随胰岛素原纤维化的结构变化（二聚体->单体->部分未折叠单体->寡聚体聚集体->原纤维）的顺序可以使用固有Tyr荧光直接检测。结果表明，至少有两个可区分的结构中间体前原纤维的发展。在胰岛素聚集过程中没有酪氨酸或二酪氨酸的证据。从蛋白质本身获得这样的关键信息是对现有聚集探针的补充，并且提供了直接检查在分子水平上发生的结构变化的优点，提供了纤颤之前的早期事件的具体细节。
The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers -> monomers -> partially unfolded monomers -> oligomeric aggregates -> fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence. The results indicate that at least two distinguishable structural intermediates precede fibril development. There is no evidence of tyrosinate or dityrosine during insulin aggregation. Obtaining such critical information from the protein itself is complementary to existing aggregation probes and affords the advantage of directly examining structural changes that occur at the molecular level, providing concrete details of the early events preceding fibrillation.