Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan
Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan
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DOI：
10.1021/bc0341324
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发表时间：
2003-11-01
影响因子：
4.7
通讯作者：
Wolfrum, J
中科院分区：
文献类型：
--
作者：
Marmé, N;Knemeyer, JP;Wolfrum, J
用稳态荧光光谱和时间分辨荧光光谱研究了恶嗪、罗丹明、碳菁染料和Bora-diaza-indacene染料对氨基酸的荧光猝灭作用。在天然氨基酸中，色氨酸表现出最显著的猝灭效率。特别是红色吸收染料ATTO 655、ATTO 680和恶嗪衍生物MR 121几乎完全被色氨酸猝灭，这是因为形成了弱的或非荧光的基态络合物，其缔合常数K-ass从96到206M-1。吸收波长较短的罗丹明、荧光素和博拉-二氮杂-吲哚衍生物也基本上被酪氨酸残基猝灭。碳菁染料如Cy5和Alexa 647被氨基酸猝灭几乎可以忽略不计。色氨酸对ATTO 655、ATTO 680和恶嗪衍生物MR121的猝灭以静态猝灭为主，而BODIPY-FL和Bodipy630/650两种Bora-diaza-indacene染料的猝灭以动态猝灭为主。对色氨酸、含色氨酸的多肽和蛋白质(链霉亲和素)的标记表明，了解这些荧光猝灭过程对于开发基于荧光的诊断分析至关重要。染料标记的多肽和蛋白质的荧光量子产率的变化可能有利于对蛋白水解酶和特定结合伙伴的定量。
Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K-ass., ranging from 96 to 206 M-1. Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.