Probing the lateral organization of membranes: fluorescence repercussions of pyrene probe distribution

Phospholipids pyrene labeled are widely used to investigate dynamics and organizations of membranes. We studied pyrene probe lateral distribution by analyzing the variations of the molar absorption coefficient (epsilon) versus probe concentrations, in small unilamellar vesicles (SUV) made of phospholipids and/or glycolipids, with pyrene labeled phosphatidylcholine (PyPC) or phosphatidylglycerol (PyPG). The results were interpreted according to an infinite associative model. They indicated that an effective self-association process corresponding to K ranging from 30 to 100 M(-1) occurred with those probes incorporated in dimannosyl diacylglycerol (DMDG). In contrast, after SUV labeling of egg yolk phosphatidylcholine (EggPC) or phosphatidylglycerol (EggPG), K values < 1 M(-1) were determined. The corresponding percentages of various stacked forms of pyrene probes were calculated. They indicated that, for a 3% PyPG labeling, the monomer represented 21% of n-mers in DMDG and 94% in EggPC. The analysis of fluorescence experiments carried out on the same samples indicated that: (i) the fluorescence process of pyrene probes was generated by the monomers: and (ii) the excimer forming resulted from a diffusional encounter between one excited and one non-excited monomer. A correction of fluorescence data allowing a more correct interpretation of fluorescence measurements was proposed.