The domain structure of cholesterol in membranes and factors affecting it are not well understood. A method, based on kinetics of delta 5,7,9,(11),22-erogostatetraen-3 beta-ol (dehydroergosterol) fluorescence polarization change and not requiring separation of donor and acceptor membranes, was used to examine sterol domains in three-component cholesterol:dehydroergosterol:phospholipid small unilamellar vesicles (SUV). A new mathematical data treatment was developed to provide a direct correlation between molecular sterol exchange and steady-state dehydroergosterol fluorescence polarization measurements. The method identified multiple kinetic pools of sterol in SUV: a small but rapidly exchanging pool, a predominant slowly exchanging pool, and a very slowly exchangeable (nonexchangeable) pool. The relative sizes of the pools and half-times of exchange were highly dependent on the presence of acidic phospholipids and on cytosolic proteins involved in sterol transfer. Thus, the method provides a direct measure of molecular sterol transfer between membranes without separating donor and acceptor membranes.
