The peptide bond quenches indole fluorescence
The peptide bond quenches indole fluorescence
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DOI：
10.1021/ja961307u
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发表时间：
1996-10-02
影响因子：
15
通讯作者：
Barkley, MD
中科院分区：
文献类型：
--
作者：
Chen, Y;Liu, B;Barkley, MD
肽键对蛋白质荧光的影响是色氨酸化学物理中一个尚未解决的重要问题。通过对一系列模型化合物的溶质猝灭研究，得到了肽基作为吲哚荧光弱猝灭剂的有力证据。需要两个酰胺用于可检测的3-甲基吲哚荧光的猝灭，并且猝灭速率取决于酰胺之间的距离。丙二酰胺、N-乙酰天冬酰胺、N-乙酰甘氨酰胺和N-乙酰谷氨酰胺的双分子速率常数k（q）分别为33 × 10（7）、8.8 × 10（7）、6.6 × 10（7）和2.2 × 10（7）M（-1）s（-1）。瞬态吸收和温度依赖性的荧光寿命测量的情况下和存在下的猝灭剂的电子转移作为猝灭机制提供了强有力的旁证。三重态产率测定五吲哚衍生物使用瞬态吸收。从三重态产率和荧光寿命数据计算系统间交叉率。系统间杂交率k（isc）从3-甲基吲哚的2.1 × 10（7）s（-1）到吲哚的7.6 × 10（7）s（-1）不等。肽基不改变3-甲基吲哚的k（isc）值。的辐射和系统间的交叉率的总和是等于3-甲基吲哚，吲哚，N-乙酰基氨基，和N-甲基吲哚的荧光衰减率的温度无关的部分，证实吲哚的系统间交叉是独立于水溶液中的温度。在N-乙酰基甘氨酰胺、乙酸乙酯和氯化钆存在下测定了3-甲基吲哚荧光寿命的温度依赖性。两个单独的Arrhenius条款解决了水淬火和溶质淬火。N-乙酰基甘氨酰胺、乙酸乙酯和GdCl 3对溶质猝灭的活化能分别为2.5 +/- 0.3、0.0和6.0 +/- 0.5 kcal/mol。对于N-乙酰基葡聚糖酰胺中肽键的分子内猝灭，活化能为3.2 +/- 0.3 kcal/mol。讨论了利用荧光寿命的温度依赖性来计算单个非辐射过程速率的策略。
The effect of the peptide bond on protein fluorescence is an important unresolved question in tryptophan photophysics. Definitive evidence for the peptide group as a weak quencher of indole fluorescence was obtained from solute quenching studies with a series of model compounds. Two amides are required for detectable quenching of 3-methylindole fluorescence and the quenching rate depends on the distance between amides. The bimolecular rate constants k(q) of malonamide, N-acetylasparagine, N-acetylglycinamide, and N-acetylglutamine are 33 x 10(7), 8.8 x 10(7), 6.6 x 10(7) and 2.2 x 10(7) M(-1) s(-1), respectively. Transient absorption and temperature dependence of the fluorescence lifetime measured in the absence and presence of quencher gave strong circumstantial evidence for electron transfer as the quenching mechanism. Triplet yields were measured for five indole derivatives using transient absorption. Intersystem crossing rates were calculated from triplet yield and fluorescence lifetime data. The intersystem crossing rate k(isc) varies from 2.1 x 10(7) s(-1) for 3-methylindole to 7.6 x 10(7) s(-1) for indole. The peptide group does not change the value of k(isc) of 3-methylindole. The sum of the radiative and intersystem crossing rates is equal to the temperature-independent portion of the fluorescence decay rate for 3-methylindole, indole, N-acetyltryptophanamide, and N-methylindole, confirming that intersystem crossing in indoles is independent of temperature in aqueous solution. The temperature dependence of the fluorescence lifetime of 3-methylindole was determined in the presence of N-acetylglycinamide, ethyl acetate, and GdCl3. Two separate Arrhenius terms were resolved for water quenching and solute quenching. The activation energies for solute quenching by N-acetylglycinamide, ethyl acetate, and GdCl3 are 2.5 +/- 0.3, 0.0, and 6.0 +/- 0.5 kcal/mol, respectively. For intramolecular quenching by the peptide bonds in N-acetyltryptophanamide, the activation energy is 3.2 +/- 0.3 kcal/mol. The strategy of using the temperature dependence of the fluorescence lifetime to calculate the rates of individual nonradiative processes is discussed.