Study of the localization of a fluorescence probe on the membrane surface using angle-resolved fluorescence depolarization method

The angle-resolved fluorescence depolarization method (AFD) has been applied to study the localization of a fluorescent probe, fluorescein isothiocyanate (FITC). FITC-labeled albumin in an aqueous glycerol solution (95%) was used as an isotropic immobilized system. The angle between absorption and emission (α) in this system was found to be 40° ± 2°. Further measurements were carried out on multibilayers from synthetic lipid dimyristoyl phosphatidylcholine (DMPC) containing FITC labeled dimyristoyl phosphatidylethanolamine, (FITC-DMPE) as a lipid probe. Experiments were performed at temperatures above (35 and 25°C) and below (17°C) the membrane phase transition temperatures. The second-rank order parameters of angular distribution of the probe in the membrane (P₂) determined at 35, 25 and 17°C were 0.13, 0.16 and 0.33, and values of angle α, 46°, 46° and 50°, respectively. Both α and (P₂) showed a significant change upon the membrane phase transition. Calculated values of the forth-rank order parameter (P₄) and rotational correlation times were found to be physically incorrect, which did not allow determining the angular distribution of the fluorescence label using the approach described in literature. This is a result of the limited applicability of the existing theoretical model to this experimental system. However, the behavior of angular dependencies of depolarization suggests that the motion is slowed down. To estimate the angular distribution of the label, numeric experiments in the absence of diffusion and at α equal to 45° were performed. The distribution was found to have maximum at 60° relative to the perpendicular to the membrane plane, with the half width of 30°. In summary, the label is oriented at a small angle to the membrane surface and does not swing considerably.