Dipolar relaxation in proteins on the nanosecond timescale observed by wavelength-resolved phase fluorometry of tryptophan fluorescence.

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.