A Fluorescence Approach of the Determination of Translational Diffusion Coefficients of Lipids in Phospholipid Monolayer at the Air-Water Interface

Abstract

In the present work, it is shown that the photobleaching technique as well as experimentation based on fluorescence recovery after bleaching can be extended to monolayers spread at the air-water interface. A mathematical model is derived which allows the determination of translational diffusion coefficients of species diffusing in such a system. Using 12-(9-anthroyl)stearic acid (anthroylstearate) as a fluorescent probe, dispersed either in dipalmitoylphosphatidylcholine or in dipalmitoylphosphatidylglycerol in various conditions of subphase ionic composition and surface pressure of the monolayer, including phase transition domains, we are led to the following conclusions :1. Anthroylstearate molecules seem to aggregate in 'microdomains' where their fluorescence properties remain unchanged regardless of the compression states of the host monolayer.2. In any case, a break in the diffusion constants appears on compressing films of both dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. In particular, this break coincides with the liquid expanded to gel phase transition of these lipids when it occurs.3. Diffusion of anthroylstearate in dipalmitoylphosphatidylglycerol depends strongly on the subphase ionic strength and on the nature of cations: Na', Mg2+, Ca2+.In recent years, much interest has been expressed in the study of lateral diffusion of phospholipids and proteins in biomembranes. Such a process which participates in the more general concept of 'membrane fluidity' seems to play a significant role in modulating membrane functions of some proteins [l]. Many of the studies reported on lateral diffusion have been carried out by use of spectroscopic methods, basically electron spin resonance [2], nuclear magnetic resonance , whereas experimentation on monolayers have been scarce and based on the use of radioactive tracers [lo]. Lipid monolayers at the air-water interface have proved to be good membrane models in which not only the temperature and subphase pH and ionic composition can be controlled, but also the molecular packing of the lipids. We have reported recently [ 111 that fluorescence measurements are feasible with such monolayers at very low concentrations of the probe (mole ratio of the order of In the present work, the photobleaching technique has been extended to monolayers and the experimentation based on fluorescence recovery after bleaching as well. A mathematical treatment of the data has been derived which allows the determination of translational diffusion coefficients in such a system. The results presented deal with the diffusion of 12-(9-anthroy1)stearic acid (anthroylstearate) dispersed either in dipalmitoylphosphatidylcholine or in dipalmitoylphosphatidylglycerol in various conditions of subphase ionic composition and surface pressure of the monolayer, including phase transition domains. 1 : 100).

A study of the structure and dynamics of complexes between polymyxin B and phosphatidylglycerol in monolayers by fluorescence

“…previously described [12], control experiments showed that the dimerization diffusion. Therefore, a fluorescence signal should be observed when illuminating the monolayer again for a short period.…”

“…The diffusion coefficients D were then obtained through a graphical resolution of the Eqn (11) [12].…”

“…Monolayers were chosen as a model membrane system because, in such a system, lipid molecular packing is well controlled and can be examined over a large surface pressure range while polymyxin B can be used in saturating conditions with respect to dipalmitoylglycerophosphoglycerol, without precipitation of the lipid. Experiments were carried out by means of an interface fluorimeter of our fabrication [l I] which allows us to measure both the lateral diffusion rate (fluorescence recovery after photobleaching experiments) [12] and the orientation [I31 of a fluorescent probe inserted in the lipid layer. Taking advantage of the axial symmetry of monolayers, a new fluorescence technique has been developed which uses linearly polarized incident beams and which can be applied for studying simultaneously both the dynamics and the orientation of the film-forming molecules.…”

Evidence for a homogeneous lateral distribution of lipids in a bacterial membrane

“…Structure and organization of biomembranes are certainly very subtle and complex and new tools are required for describing the membrane assembly at a microscopic scale, both in terms of organization and dynamics of the various components. Remembering that anthracene is fluorescent and that its photo-dimers are not [lo], it is worth noting that this group can be used not only for topological studies of lipids in membranes but also for investigating their lateral diffusion rate [22,23].…”

Metabolic incorporation of 9-(2-anthryl)-nonanoic acid, a new fluorescent and photoactivable probe, into the membrane lipids of Chinese hamster ovary cells

“…This hydrophobic group, which is well suited for labelling the hydrophobic core of the membrane, is fluorescent and under illumination (360 nm) it forms 9-9', 10-10 covalently bound dimers, which are no longer fluorescent [19]. Thus, after its incorporation into the membrane lipids, this group has potential uses (a) as an indicator of membrane fluidity [20]; (b) for measuring the lateral diffusion rate of the labelled molecules by using the technique of fluorescence recovery after the photodimerization reaction [21] ; (c) for studying the topological distribution of lipids in membranes by means of a photo-cross-linking reaction between adjacent anthracene-labelled molecules (dimerization) and identification of the photodimers.…”