The sensitivity of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) excitation and emission spectra to the physical state of the membrane arises from dipolar relaxation processes in the membrane region surrounding the Laurdan molecule. Experiments performed using phospholipid vesicles composed of phospholipids with different polar head groups show that this part of the molecule is not responsible for the observed effects. Also, pH titration in the range from pH 4 to 10 shows that the spectral variations are independent of the charge of the polar head. A two-state model of dipolar relaxation is used to qualitatively explain the behavior of Laurdan. It is concluded that the presence of water molecules in the phospholipid matrix are responsible for the spectral properties of Laurdan in the gel phase. In the liquid crystalline phase there is a relaxation process that we attribute to water molecules that can reorientate during the few nanoseconds of the excited state lifetime. The quantitation of lipid phases is obtained using generalized polarization which, after proper choice of excitation and emission wavelengths, satisfies a simple addition rule.
Coexisting gel and liquid-crystalline phospholipid phase domains can be observed in synthetic phospholipid vesicles during the transition from one phase to the other and, in vesicles of mixed phospholipids, at intermediate temperatures between the transitions of the different phospholipids. The presence of cholesterol perturbs the dynamic properties of both phases to such an extent as to prevent the detection of coexisting phases. 6-Lauroyl-2-dimethylaminopahthalene (Laurdan) fluorescence offers the unique advantage of well resolvable spectral parameters in the two phospholipid phases that can be used for the detection and quantitation of coexisting gel and liquid-crystalline domains. From Laurdan fluorescence excitation and emission spectra, the generalized polarization spectra and values were calculated. By the generalized polarization phospholipid phase domain coexistence can be detected, and each phase can be quantitated. In the same phospholipid vesicles where without cholesterol domain coexistence can be detected, above 15 mol% and, remarkably, at physiological cholesterol concentrations, > or = 30 mol%, no separate Laurdan fluorescence signals characteristic of distinct domains can be observed. Consequences of our results on the possible size and dynamics of phospholipid phase domains and their biological relevance are discussed.
The effect of cholesterol on the gel, the liquid-crystalline, and mixed phospholipid phases has been studied using the fluorescence properties of 2-dimethylamino-6-lauroylnaphthalene (Laurdan). Laurdan sensitivity to the polarity and to the dynamics of its environment reveals that cholesterol affects phospholipid bilayers in the gel phase by expelling water and by increasing the amount of dipolar relaxation. In the liquid-crystalline phase, the effect of cholesterol is a reduction of both water concentration and amount of dipolar relaxation. Detailed studies of Laurdan excitation and emission spectral contours as a function of cholesterol concentration show that there are some cholesterol concentrations at which Laurdan spectral properties changes discontinuously. These peculiar cholesterol concentrations are in agreement with recent observations of other workers showing the formation of local order in the liquid-crystalline phase of phospholipids upon addition of phospholipid derivatives of pyrene. A local organization of phospholipids around cholesterol molecule seems to be produced by the presence of peculiar concentrations of cholesterol itself. This local organization is stable enough to be observed during the excited state lifetime of Laurdan of approximately 5-6 ns.
Abstract-Steady-state and dynamic fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) have been used to ascertain the coexistence of separate phase domains and their dynamic properties in phospholipid vesicles composed of different mole ratios of dilauroyl-and dipalmitoyl-phosphatidylcholine (DLPC and DPPC, respectively). The recently introduced generalized polarization together with time-resolved emission spectra have been utilized for detecting changes. The results indicate the coexistence of phospholipid phase domains in vesicle compositions in the range between 30 mol% and 70 mol% DPPC in DLPC. Below and above these concentrations a homogeneous phase is observed, with averaged properties. In the case of coexisting phase domains, the properties ofeach individual phase are largely influenced by the presence ofthe other phase. Implications on fluctuations between the coexisting phases and on the size and shape of domains are discussed.
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