Membrane Heterogeneity in Erythrocytes Studied by Laurdan GP and Flim-Phasor

Detergents can selectively solubilize membrane constituents by incorporating them into small micelles, whereas other components may remain in the so-called detergent-resistant membrane fragments. This differential interaction provides a unique tool to selectively isolate proteins and study their localization into specific membrane environments or domains. Laurdan Generalized Polarization (Laurdan GP), defined as [(I440-I490)/(I440+I490)] (1), measures the red spectral shift of the molecule due to polarity and dipole relaxation processes. This parameter gives valuable information about membrane heterogeneity; however there is more information veiled behind the Laurdan spectral shift that can be revealed by lifetime measurements at the two GP wavelengths (2). Lifetime measurements can isolate the two effects measured by Laurdan GP: the polarity of the environment (reported mainly by the intensity at 440 nm) and the rate of dipolar relaxation of water molecules that can reorient around laurdan's dipole during its lifetime (monitored at 490 nm)We used sucrose monoester of myristic acid, b-D-Fructofuranosyl-6-O-myristyl-a-D-Glucopyranoside (MMS) and Laurdan (6-lauroyl,1-2-dimethylamino naphthalene) to study the changes promoted in membrane heterogeneity in erythrocytes with different cholesterol content. We followed the hemolysis, the changes in size and the changes in fluidity on the membrane of erythrocytes treated with increasing amount of MMS. Our results indicate that the insertion of MMS molecules in the membrane produces a decrease in polarity of the bilayer (reported mainly by the intensity at 440 nm) together with an increase on dipolar relaxation of water molecules around Laurdan (reported at 490 nm).Financial support: Fondecyt #1140454 (S.S.), Fondecyt# 1120196 (G.G.) and Beca de Doctorado Conicyt 21120554 (C.S.). Technical Support: CMABioBio-UdeC.[1] Parasassi T, et al. Biophys J. 57; 1179, 1990.[2] Golfetto O, et al. Biophys J. 104, 1238-1247, 2013