Lipid peroxides promote large rafts: effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation

¹ Rush University Medical Center

^* To whom correspondence should be addressed. E-mail: fcohen{at}rush.edu.

Submitted on April 18, 2006
Revised on May 25, 2006
Accepted on 8 June 2006

	Abstract

Raft formation and enlargement was investigated in liposomes and supported bilayers prepared from sphingomyelin (SM), cholesterol, and unsaturated phospholipids; NBD-DPPE and Rhodamine-DOPE were employed as fluorescent probes. Rafts were created by lowering temperature. Maintaining 20 mol% SM, fluorescence microscopy showed that, in the absence of photo-oxidation, large rafts did not form in GUVs containing 20 or more mol% cholesterol. But if photo-oxidation was allowed to proceed, large rafts were readily observed. In population, cuvette experiments, small rafts formed without photo-oxidation at high cholesterol concentrations. Thus, photo-oxidation was the cause of raft enlargement during microscopy experiments. Because photo-oxidation results in peroxidation at lipid double bonds, photosensitization experiments were performed to explicitly produce peroxides of SM and an unsaturated phospholipid. GUVs of high cholesterol content containing the breakdown products of SM-peroxide, but not phospholipid-peroxide, resulted in large rafts after lowering temperature. In addition, GUV production by electroswelling can result in peroxides that cause large raft formation. The use of titanium electrodes eliminates this problem. In conclusion, lipid peroxides and their breakdown products are the cause of large raft formation in GUVs containing biological levels of cholesterol. It is critical that experiments investigating rafts in bilayer membranes avoid the production of peroxides.

Key Words: FRET, Fluorescence microscopy, Giant unilamellar vesicles, cushioned bilayers, lipid oxidation, supported bilayers

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