Demonstration of an associated anisotropy decay by frequency-domain fluorometry

We used frequency-domain fluorometry to demonstrate the presence of an associated decay of fluorescence anisotropy. In such systems the individual correlation times are associated with distinct emitting species, each with its own characteristic lifetime and rotational correlation times. We obtained an associated system using 1-anilino-8-naphthalenesulfonic acid (ANS) in the presence of increasing amounts of apomyoglobin. When both free and apomyoglobin-bound ANS contributed to the emission the differential polarized phase angles become negative at particular frequencies, even though the fundamental anisotropy (r0) is greater than zero. Additionally, the modulated anisotropy decreases at high frequencies. Both observations appear to be the unique consequence of an associated anisotropy decay, and are not possible for a multiexponential anisotropy decay of a single species.