Lakowicz J R, Gryczynski I, Cherek H, Laczko G
University of Maryland at Baltimore, Center for Fluorescence Spectroscopy and School of Medicine, Department of Biological Chemistry, 660 West Redwood Street, Baltimore, MD 21201, USA.
Biophys Chem. 1991 Mar;39(3):241-51. doi: 10.1016/0301-4622(91)80002-9.
We used frequency-domain fluorescence spectroscopy to measure the fluorescence lifetime and anisotropy decays of indole in propylene glycol, and of the tryptophan emission of melittin monomer and tetramer in water solutions at 5 degrees C. We obtained an increase in resolution of the anisotropy decays by using multiple excitation wavelengths, chosen to provide a range of fundamental anisotropy values. The multi-excitation wavelength anisotropy decays were analyzed globally to recover a single set of correlation times with wavelength-dependent anisotropy amplitudes. Simulated data and kappaR2 surfaces are shown to reveal the effect of multi-wavelength data on the resolution of complex anisotropy decays. For both indole and melittin, the anisotropy decays are heterogeneous and require two correlation times to fit the frequency-domain data. For indole in propylene glycol at 5 degrees C we recovered correlation times of 0.59 and 4.10 ns, which appear to be characteristic of the rigid and asymmetric indole molecule. For melittin monomer the correlation times were 0.13 and 1.75 ns, and for melittin tetramer 0.12 and 3.96 ns. The shorter and longer correlation times of melittin are due to segmental motions and overall rotational diffusion of the polypeptide.
我们使用频域荧光光谱法测量了吲哚在丙二醇中的荧光寿命和各向异性衰减，以及蜂毒肽单体和四聚体在5摄氏度水溶液中的色氨酸发射荧光的各向异性衰减。我们通过使用多个激发波长提高了各向异性衰减的分辨率，这些激发波长的选择是为了提供一系列基本各向异性值。对多激发波长各向异性衰减进行全局分析，以恢复一组与波长相关的各向异性幅度的单一相关时间。展示了模拟数据和kappaR2表面，以揭示多波长数据对复杂各向异性衰减分辨率的影响。对于吲哚和蜂毒肽，各向异性衰减都是非均匀的，需要两个相关时间来拟合频域数据。对于5摄氏度丙二醇中的吲哚，我们恢复的相关时间为0.59和4.10纳秒，这似乎是刚性且不对称的吲哚分子的特征。对于蜂毒肽单体，相关时间为0.13和1.75纳秒，对于蜂毒肽四聚体，相关时间为0.12和3.96纳秒。蜂毒肽较短和较长的相关时间分别归因于多肽的片段运动和整体旋转扩散。
