Measuring rotational diffusion of macromolecules by fluorescence correlation spectroscopy.

We describe a novel method to measure rotational diffusion of large biomolecules in solution based on fluorescence correlation on the nanosecond time scale. In contrast to conventional fluorescence anisotropy measurements, a correlation-based method will also work if the rotational diffusion time is much longer than the fluorescence decay time. Thus, the method is suited to study the rotational diffusion of macromolecules having rotational diffusion times of dozens to hundreds of nanoseconds, which is considerably larger than the fluorescence lifetime of most commercially available dyes or auto-fluorescent proteins. A pulsed interleaved excitation scheme with crossed excitation polarization maximizes the time-dependent amplitude of the measured correlation curve as caused by rotational diffusion. Using the determined rotational diffusion coefficient, precise values of the hydrodynamic radius can be obtained. The method is exemplified on sizing a set of common globular proteins.