Molecular movement fluorescence depolarization

Limpouchova Z, Prochazka K, Fidler V, Dvorak J, Bednar B (1993) Molecular-movements and dynamics in solutions studied by fluorescence depolarization measurement. Collect Czech Chem Commun 58(2) 213-233. doi 10.1135/ccccl9930213... 

Fluorescence anisotropy measurements can also be used to obtain the rates of the excited state tautomerization. Two variants can be applied. The first is based on the analysis of time-resolved anisotropy curves. These are constructed from measurements of the fluorescence decay recorded with different positions of the polarizers in the excitation and emission channels. The anisotropy decay reflects the movement of the transition moment and thus, the hydrogen exchange. For molecules with a long-lived Sj state, the anisotropy decay can also be caused by rotational diffusion. In order to avoid depolarization effects due to molecular rotation, the experiments should be carried out in rigid media, such as polymers or glasses. When the Sj lifetime is short compared to that of rotational diffusion, tautomerization rates can be determined even in solution. This is the case for lb, for which time-resolved anisotropy measurements have been performed at 293 K, using a... 

We have consistently talked about the polarization of a fluor bound to a macromoleeule. What about the intrinsic fluorescence of the macromolecules and its polarization For example the intrinsic fluorescence of tryptophan in a protein and utilization of its polarization in stud3dng the protein. There is a problem here. Large proteins move very slowly on a molecular scale. Thus, to observe depolarization due to motion, the lifetime of the excited state should be sufficiently long, i.e., there should be a good time lag between excitation and emission so that the molecule may show substantial movement in that time and depolarization may occur. For very small proteins, intrinsic fluorescence may be of some use, but for larger proteins, extrinsic fluorescence has to be made use of. 

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