Ross J B, Schmidt C J, Brand L
Biochemistry. 1981 Jul 21;20(15):4369-77. doi: 10.1021/bi00518a021.
The tryptophan fluorescence decay of horse liver alcohol dehydrogenase, at 10 degrees C in 0.1 M pH 7.4 sodium phosphate buffer, with excitation at 295 nm, is a double exponential with time constants of 3.8 and 7.2 ns. Within experimental error, the two lifetimes remain constant across the emission spectrum. Only the 3.8-ns lifetime is quenched in the NAD+-pyrazole ternary complex, and only the 7.2-ns lifetime is quenched by 0-0.05 M KI. On the basis of these results, we assign the 3.8-ns lifetime to the buried tryptophan, Trp-314, and the 7.2-ns lifetime to the exposed tryptophan, Trp-15. The steady-state lifetime-resolved emission spectrum of Trp-15 has a maximum at approximately 340 nm and that of Trp-15 is at approximately 325 nm. The total time-resolved emission, after 40 ns of decay, has a maximum between 338 and 340 nm and is primarily due to the Trp-15 emission. As a consequence of the wavelength dependence of the preexponential weighting factors, there is an increase in the average lifetime from the blue to the red edge of the emission. This increase reflects the change in the spectral contributions of Trp-15 and Trp-314. Consideration of the spectral overlap between the emission spectra of the two tryptophans and the absorption due to formation of the ternary complex, as well as the distances between the two residues and the bound NAD+, shows that the selective fluorescence quenching in the ternary complex can be accounted for entirely by singlet-single energy transfer. The decay of the fluorescence anisotropy was measured as a function of temperature from 10 to 40 degrees C and is well described by a monoexponential decay law. Over this temperature range the calculated hydrodynamic radius increases from 33.5 to 35.1 A. Evidently, the indole groups of Trp-15 and Trp-314 rotate with the protein as a whole, and there is some expansion of the protein matrix as the ambient temperature is increased.
在0.1M pH 7.4的磷酸钠缓冲液中，于10℃下，对马肝醇脱氢酶进行色氨酸荧光衰减实验，激发波长为295nm，其荧光衰减呈双指数形式，时间常数分别为3.8ns和7.2ns。在实验误差范围内，这两个寿命在整个发射光谱中保持恒定。在NAD⁺ - 吡唑三元复合物中，只有3.8ns的寿命被猝灭，而在0 - 0.05M KI存在时，只有7.2ns的寿命被猝灭。基于这些结果，我们将3.8ns的寿命归属于埋藏的色氨酸Trp - 314，将7.2ns的寿命归属于暴露的色氨酸Trp - 15。Trp - 15的稳态寿命分辨发射光谱在约340nm处有最大值，Trp - 314的在约325nm处。在40ns的衰减后，总的时间分辨发射在338至340nm之间有最大值，并且主要归因于Trp - 15的发射。由于预指数加权因子的波长依赖性，从发射光谱的蓝端到红端，平均寿命增加。这种增加反映了Trp - 15和Trp - 314光谱贡献的变化。考虑到两个色氨酸发射光谱之间的光谱重叠以及三元复合物形成引起的吸收，以及两个残基与结合的NAD⁺之间的距离，表明三元复合物中的选择性荧光猝灭完全可以由单重态 - 单重态能量转移来解释。荧光各向异性的衰减作为温度从10℃到40℃的函数进行测量，并且由单指数衰减定律很好地描述。在这个温度范围内，计算出的流体动力学半径从33.5Å增加到35.1Å。显然，Trp - 15和Trp - 314的吲哚基团与整个蛋白质一起旋转，并且随着环境温度升高，蛋白质基质有一些膨胀。
