Time‐dependent fluorescence depolarization and Brownian rotational diffusion coefficients of macromolecules

Abstract Using a distribution function formalism, we have derived relations between the time‐dependent fluorescence polarization anistropy r ( t ) = I ‖( t ) − I ⟂( t )/ I ‖( t ) + 2 I ⟂( t ) and the Brownian rotational diffusion coefficients of macromolecules. It is shown that in the most general case of a completely asymmetric body, five exponentials appear in r ( t ). Reduction to previously obtained simpler expressions are presented and discussed. Experiments were performed to measure r ( t ) for the complex of 1‐anilino‐8‐naphthalene sulfonate (ANS) and apomyoglobin. The results revealed a spherical geometry, with a radius of 20.8 Å. Similar experiments were performed on the ANS–apohorse radish peroxidase complex. The results were inconclusive towards the geometry of the complex, but when assumed to be spherical, the radius was estimated to be 29.6 Å, consistent with the larger molecular weight of horse radish peroxidase.