Lakowicz J R, Cherek H
J Biol Chem. 1980 Feb 10;255(3):831-4.
We used phase fluorometry to investigate the wavelength dependence of the fluorescence lifetimes of N-acetyl-L-tryptophanamide (NATA) in solvents of varying viscosity and the lifetimes of tryptophan in human serum albumin, melittin, and liver alcohol dehydrogenase. In highly fluid solvents, and in completely vitrified solvents, the lifetime of NATA was constant across its emission spectrum. In viscous solvents, such as propylene glycol at -9 degrees C, the lifetimes of NATA increased across its emission spectrum, with the values being 3.3, 5.5, and 8.1 ns at 317, 344, and 400 nm, respectively. These wavelength-dependent lifetimes appear to be a result of reorientations of solvent dipoles around the excited state dipole moment of the indole moiety. For the three proteins investigated, the fluorescence lifetimes of tryptophan increased with increasing wavelength in a manner comparable to that observed for NATA in propylene glycol. These observations indicate that these protein matrices can reorientation around their tryptophan residues on the nanosecond timescale, and illustrate the potential of phase fluorometry for quantifying the details of these dipolar relaxation processes.
我们使用相荧光测定法研究了在不同粘度溶剂中N-乙酰-L-色氨酸酰胺（NATA）荧光寿命的波长依赖性，以及在人血清白蛋白、蜂毒肽和肝醇脱氢酶中色氨酸的荧光寿命。在高流动性溶剂和完全玻璃化的溶剂中，NATA在其发射光谱范围内的寿命是恒定的。在粘性溶剂中，如-9℃的丙二醇，NATA在其发射光谱范围内的寿命增加，在317、344和400nm处的值分别为3.3、5.5和8.1ns。这些波长依赖性寿命似乎是溶剂偶极围绕吲哚部分激发态偶极矩重新取向的结果。对于所研究的三种蛋白质，色氨酸的荧光寿命随波长增加，其方式与在丙二醇中观察到的NATA相似。这些观察结果表明，这些蛋白质基质可以在纳秒时间尺度上围绕其色氨酸残基重新取向，并说明了相荧光测定法在量化这些偶极弛豫过程细节方面的潜力。
