Abstract
2-Dimethylamino-6-lauroylnaphthalene (Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. Steady-state Laurdan excitation and emission spectra have different maxima and shape in the two phospholipid phases, due to differences in the polarity and in the amount of dipolar relaxation. In bilayers composed of a mixture of gel and liquid-crystalline phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases. These spectral properties are analyzed using the generalized polarization (GP). TheGP value can be used for the quantitation of each phase. The wavelength dependence of theGP value is used to ascertain the coexistence of different phase domains in the bilayer. Moreover, by following the evolution of Laurdan emission vs. time after excitation, the kinetics of phase fluctuation in phospholipid vesicles composed of coexisting gel and liquid-crystalline phases was determined.GP measurements performed in several cell lines did not give indications of coexistence of phase domains in their membranes. In natural membranes, Laurdan parameters indicate a homogeneously fluid environment, with restricted molecular motion in comparison with the phospholipid liquid-crystalline phase. The influence of cholesterol on the phase properties of the two phospholipid phases is proposed to be the cause of the phase behavior observed in natural membranes. In bilayers composed of different phospholipids and various cholesterol concentrations, Laurdan response is very similar to that arising from cell membranes. In the absence of cholesterol, from the steady-state and time-resolved measurements of Laurdan in phospholipid vesicles, the condition for the occurrence of separate coexisting domains in the bilayer has been determined: the molecular ratio between the two phases must be in the range between 30% and 70%. Below and above this range, a single homogeneous phase is observed, with the properties of the more concentrated phase, slightly modified by the presence of the other. Moreover, in this concentration range, the calculated dimension of the domains is very small, between 20 and 50 Å.
Highlights
2-Dimethylamino-6-1auroylnaphthalene (Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment
Steady-state Laurdan excitation and emission spectra have different maxima and shape in the two phospholipid phases, due to differences in the polarity and in the amount of dipolar relaxation, ha bilayers composed of a mixture of gel and liquid-crystalline phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases
Additional experimental problems prevented the use of DPH for the detection and the resolution of coexisting domains in membranes: (i) The difference in the average lifetime values measured in each pure phase is small. (ii) DPH decay in phospholipid vesicles as a function of temperature can be described by a linear superposition of the properties arising from the two phases or by a continuous variation of the properties of the probe along the phase transition
Summary
Received February 16, 1994; revised September 27, 1994; accepted September 27, 1994. 2-Dimethylamino-6-1auroylnaphthalene (Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. In the absence of cholesterol, from the steady-state and time-resolved measurements of Laurdan in phospholipid vesicles, the condition for the occurrence of separate coexisting domains in the bilayer has been determined: the molecular ratio between the two phases must be in the range between 30% and 70%. And above this range, a single homogeneous phase is observed, with the properties of the more concentrated phase, slightly modified by the presence of the other. Phase domains could create separate compartments for the different membrane activities, each requiring peculiar local dynamical properties
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