Fluorescence Polarization Studies with and without Time Resolution

Fluorescence Polarization Studies with and without Time Resolution 

If the aromatic group is bound tightly within the protein molecule, then one may obtain information on the rotational diffusion of the whole molecule from fluorescence polarization studies. Such investigations, which were started by Weber,(68) were widely popular in the 1960s and 1970s. Correlation times t D of macromolecule rotations were determined according to the Perrin equation  

The elucidation of the intramolecular dynamics of tryptophan residues became possible due to anisotropy studies with nanosecond time resolution. Two approaches have been taken direct observation of the anisotropy kinetics on the nanosecond time scale using time-resolved(28) or frequency-domain fluorometry, and studies of steady-state anisotropy for xFvarying within wide ranges (lifetime-resolved anisotropy). The latter approach involves the application of collisional quenchers, oxygen(29,71) or acrylamide.(30) The shortening of xF by the quencher decreases the mean time available for rotations of aromatic groups prior to emission. 

In order to avoid complications caused by excitation energy transfer between tryptophan residues, most investigations have been performed with proteins containing one tryptophan residue per molecule. When studying protein solutions, there are difficulties in separating the effects of rotation of entire protein molecules and of the chromophores themselves relative to their environment in the protein matrix. It is usually assumed that intramolecular motions are more rapid and manifest themselves as short-lived components of anisotropy decay curves or in depolarization at short emission lifetimes. 

Recent results show large variations in intramolecular rotations of tryptophan residues in proteins on the nanosecond time scale, ranging from complete absence of mobility to motions of considerable angular amplitudes. Among native proteins with internal tryptophan residues, wide angular amplitude rotations were observed only in studies of azurin,(28 29) where the correlation time of the rapid component was x = 0.51 ns.(28) The existence of 

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