New Insights on the Behavior of PRODAN in Homogeneous Media and in Large Unilamellar Vesicles Fernando Moyano, M. Alicia Biasutti, Juana J. Silber,* and N. Mariano Correa* Departamento de Quı ´mica, UniVersidad Nacional de Rı ´o Cuarto, Agencia Postal # 3, X5804ZAB Rı ´o Cuarto, Argentina ReceiVed: December 9, 2005; In Final Form: March 30, 2006 The behavior of 6-propionyl-2-dimethylaminonaphthalene (PRODAN) was studied in homogeneous media and in large unilamellar vesicles (LUVs) of the phospholipid 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine (DOPC), using absorption, emission, depolarization, and time-resolved spectroscopies. In homogeneous media, the Kamlet and Taft solvatochromic comparison method quantified solute-solvent interactions from the absorption and emission PRODAN bands. These studies demonstrate that the absorption band is sensitive to the polarity-polarizability (π*) and the hydrogen bond donor ability (R) parameters of the media. PRODAN in the excited state is even more sensitive to these parameters and to the hydrogen bond acceptor ability () of the media. The transition energy (expressed in kcal/mol) for both absorption and emission bands gives a linear correlation with the well-known polarity parameter E T(30) . The results from the absorption and emission bands also reveal that PRODAN aggregates in water. The monomer has two fluorescence lifetimes, 2.27 and 0.65 ns, while the aggregate has a lifetime of 14.6 ns. Using steady-state anisotropy measurements, the calculated volumes of the aggregate and the monomer are 5590 and 222 mL mol -1 , respectively. In DOPC LUVs, PRODAN undergoes a partition process between the water bulk and the DOPC bilayer. We show that the partition constant (K p ) value is large enough that only at [DOPC] below 0.15 mg/mL PRODAN in water can be detected. PRODAN dissolved in LUVs at [DOPC] > 1 mg/mL exists completely incorporated in its monomer form and senses two different microenvironments within the bilayer: a polar region in the interface near the water and a less polar and also less viscous environment, between the phospholipid tails. These environments were characterized by their fluorescence lifetimes (τ), showing that PRODAN in the polar microenvironment has a τ value of approximately 4 ns while in the less polar region gives a value of 1.2 ns. Moreover, this probe also senses the micropolarity of these two different regions of the bilayer and yields values similar to that of methanol and tetrahydrofuran. Introduction Organized molecular assemblies, such as vesicles or lipo- somes, can be considered as large cooperative units with very different characteristics from the individual structural units which constitute them. 1 Phospholipids are the fundamental matrix of natural membranes and represent the environment in which many proteins and enzymes display their activity. 2 The problems associated with the wide diversity in composition and structure of biological membranes are avoided if one uses synthetic liposomes or vesicles which mimic the geometry, topology, and skeletal structure of cell membranes but lack ion channels and the multitude of other embedded components such as proteins. 3-7 Results using small unilamellar vesicles (SUVs) as model systems for cells are often controversial because of their size and bilayer defects due to the high curvature. For this reason, it is common to use large unilamellar vesicles (LUVs) to get closer to cell-like structures. 8 On the other hand, interactions of small molecules with membranes are important issues in membrane biology. Under- standing their role in modulating the structure and function of biological membranes requires knowledge of the location of the molecules and the degree of perturbation that it may cause in them. Many aspects of this subject were investigated in model membrane systems using different spectroscopic techniques in order to characterize the membrane structure and dynamics. 9-11 Fluorescence spectroscopy has several advantages including a high sensitivity, a noninvasive nature, an intrinsic time scale, and an excellent response to the physical properties of the membrane. 1,12,13 In general, for a fluorophore in a bulk no viscous solvent, the dipolar relaxation of the solvent molecules around its excited state is much faster than the fluorescence lifetime. In this way, the wavelength of maximum emission usually is independent of the excitation wavelength when only one species and an S 0 f S 1 transition are involved. However, excitation wavelength dependence is observed if the dipolar relaxation of the solvent molecules is slow in the excited state, such that the relaxation time is comparable to or longer than the fluorescence lifetime. Such a shift in the wavelength of maximum emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is known as the red-edge excitation shift (REES). This effect is mostly observed with polar fluorophores, whose motion is restricted by media such as very viscous solutions or condensed phases. 1,14-17 Accordingly, the REES can serve as an indicator of the fluorophore microenvironment. It is well-known that 6-propionyl-2-dimethylaminonaphtha- lene (PRODAN) is a probe particularly sensitive to the polarity * To whom correspondence should be addressed. E-mail: jsilber@ exa.unrc.edu.ar (J.J.S.); mcorrea@exa.unrc.edu.ar (N.M.C.). 11838 J. Phys. Chem. B 2006, 110, 11838-11846 10.1021/jp057208x CCC: $33.50 © 2006 American Chemical Society Published on Web 05/28/2006