Packing of phospholipid vesicles studied by oxygen quenching of Laurdan fluorescence

Abstract

Steady-state fluorescence oxygen quenching experiments were performed on phospholipid vesicles where 2-dimethylamino-6-1auroylnaphthalene (Laurdan) was inserted. The quenching efficiency was found to be much higher in vesicles in the liquid-crystalline phase with respect to the gel phase, by a factor of about 50. Since the oxygen solubility in the two phospholipid phases can differ at most by a factor of 4 based on literature values, we concluded that oxygen diffusion must be responsible for the great difference in the quenching efficiency. A relatively high quenching efficiency was also found in vesicles composed of equimolar gel and liquid-crystalline phospholipids. Simulations were performed using the linear superposition of the properties of the pure phases to demonstrate that, in the case of vesicles composed of coexisting phases, the diffusional properties of oxygen in each phase are largely modified by the presence of the other. The addition of 10 mol% cholesterol to the gel phase rendered Laurdan fluorescence approximately as quenchable as in the equimolar mixture of the two phases. This result points out that molecules such as cholesterol, which introduce packing defects in the bilayer, favor oxygen diffusion. From the oxygen quenching experiments and using the properties of generalized polarization, the rate of Laurdan dipolar relaxation can be estimated.

Membrane lipid domains and dynamics as detected by Laurdan fluorescence

“…In our calculation of emission GP, the intensities of the exci- [30]. The use of the GP offers several advantages: (i) once characteristic values for the GP in the gel and in the liquid-crystalline phase are determined, the property of additivity of fluorescence polarization can be used to quantitate the domains [27]; (ii) all properties of fluorescence polarization can be used, among others the possibility of determining the kinetics of dipolar relaxation by an equation equivalent to the Perrin equation [27,30,31]; (iii) in contrast to common ratiometric measurements used for probes displaying spectral sensitivity to the properties of the environment, such as pH and calcium concentration probes, the GP measurement does not require calibration; and (iv) the measurement is fast and easy.…”

Cholesterol protects the phospholipid bilayer from oxidative damage

“…A large difference, of about a factor of 50, between oxygen diffusion in the gel and in the liquid-crystalline phospholipid phases has been found. 8 The tight packing of phospholipids in the gel phase can be perturbed easily due to the presence of molecules different from phospholipids. For example, the addition of 10 mol% cholesterol to the gel phase renders the oxygen diffusion quite similar to that determined in a 50% mixture of the gel and liquid-crystalline phases.…”

“…For example, the addition of 10 mol% cholesterol to the gel phase renders the oxygen diffusion quite similar to that determined in a 50% mixture of the gel and liquid-crystalline phases. 8 In the present communication, we report results obtained using the above techniques, both the determination of the fluorescence decay with the harmonic response technique and the measurement of oxygen quenching of the fluorescence intensity, for the study of the damage produced by ionizing radiation on phospholipid membranes. The bilayers are composed of unsaturated phospholipids and cholesterol.…”

Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types

“…In addition, a GP value can also be obtained by measuring the relative intensities of the two excitation bands associated with the two phases, when observed at one of the two emission wavelengths characteristic of each phase. For the possibility of selective excitation of different populations of the probe and for the possibility of utilizing the properties of fluorescence polarization, Laurdan GP measurement is of extreme potential interest [18][19][20].…”