A spectroscopic technique for measuring slow rotational diffusion of macromolecules. 2: Determination of rotational correlation times of proteins in solution.

Experiments have been performed to test the validity of measurements of slow rotational diffusion using eosin isothiocyanate as a probe. When eosin-protein conjugates are covalently bound to Sepharose 4B, the dichroism of the flash-induced absorption transient does not decay with time. The magnitude of the dichroism is smaller than for eosin in solid solution. These results, together with measurements with highly viscous solutions, demonstrate the existence of a rapid but restricted independent motion of the eosin probe. This causes a partial loss of pf dochroism leaving a residual dichroism whose time dependence is determined by the rotational motion of the protein. Rotational correlation times in the microsecond-millisecond time range have been determined for eosinprotein conjugates dissolved in glycerol-water mixtures of varying viscosity. The measured values are in good agreement with theoretical predictions and show the correct dependence on the viscosity of the medium and the size and state of association of the protein.