Fluorescence depolarization by anisotropic rotational diffusion of a luminophore and its carrier molecule

The analytical expression for the polarization anisotropy is derived for a luminophore undergoing rotational diffusion about a single axis while attached to a nonluminescing, rotationally diffusing, symmetrical carrier molecule. In contrast to previous related calculations, the rotation axis of the luminophore is assumed to have an arbitrary orientation relative to the carrier. Additionally, the polarization anisotropy is measured for bovine serum albumin (BSA) labeled with dansyl, NBD, rhodamine, or eosin that is: (a) surface adsorbed to a glass/buffer interface, using a variation of the technique of total internal reflection fluorescence spectroscopy (TIRFS), or (b) bulk dissolved, using conventional transmitted illumination fluorescence spectroscopy. With this theory, using previously published values for the rotational diffusion constants of BSA and the fluorescence lifetimes of the fluorophores, the rotational diffusion constant of the covalently bound probes is estimated from the measured anisotropy values. The results indicate a wide variability in the rotational diffusion constant of the probes (from ∼10⁷ s^-1 for dansyl to ∼10⁹ s ^-1 for eosin) attached to both the surface adsorbed and bulk dissolved forms of BSA. Contrasting the rotational diffusion constant for each probe for surface adsorbed BSA vs bulk dissolved BSA indicates surface adsorption of the BSA molecule inhibits the rotational motion of the probe. These results have important implications in the application of other fluorescence techniques, such as singlet-singlet energy transfer, where the rotational mobility of the probe is important.