Photo-induced dimerization of anthracene phospholipids for the study of the lateral distribution of lipids in membranes.

Abstract

It is shown that photo-induced cross-linking reaction between anthracene-labelled phospholipids can be used for studying, at a molecular level, their lateral distribution in bilayer structures. A simple and versatile method is proposed. It is based on the property of anthracene to form covalently bound dimers upon irradiation in the near ultraviolet (360 nm) and on the possibility of separating the lipid photo-dimers from the lipid monomers by thin-layer chromatography. Identification of the photo-dimers is easily achieved since, upon illumination at a shorter wavelength (250-280 nm), they partially dissociate to the native monomer molecules. The feasibility of the method was tested by checking the effects of cations (sodium, calcium) on the homogeneity of 1/1 mixtures of anthracene-phosphatidylcholine, i.e. 1-acyl-2-[9-(2-anthryl)-nonanoyl]-sn-glycero-3-phosphocholin e (Anthr-PC) with anthracene-phosphatidic acid (Anthr-PA) and with anthracene-phosphatidylglycerol (Anthr-PG) in the form of liposomes. These lipids were anthracene-labelled by acylation of their glycerol backbone at the sn-2 position with the synthetic 9-(2-anthryl)-nonanoic acid. Data presented indicate a good miscibility of these lipids in the presence of sodium. For each lipid mixture, the lipid heterodimers were clearly identified and, quantitatively, they dominated the lipid homodimers, as expected for a regular distribution of the lipids in the 1/1 mixture. Addition of calcium ions to the lipid suspensions did not alter the miscibility properties of Anthr-PC and Anthr-PG. In contrast, calcium triggered a clear-cut phase separation in the Anthr-PC/Anthr-PA mixture as, in this case, only traces of the heterodimer form of the lipids remained observable on the chromatogram. The three anthracene-phospholipids, pure or mixed together, exhibit a clear-cut gel-to-liquid phase transition which was detectable by fluorescence intensity measurements. The analysis of the corresponding phase-transition temperatures confirms, at a 'macroscopic' level, the effects of sodium and calcium on the mixing properties of the anthracene phospholipids which were revealed at a 'microscopic' level by the dimerization procedure.