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Characterization of fatty acid vesicles by EPR

Vesicles are considered as reasonable models for protocells, the precursors of the first cells that might appeare during the prebiological evolution1. The simplest vesicles can be formed from fatty acids if approximately half of their carboxylic groups are ionised2. Since they are chemically simple and have been found in carbonaceous meteorites3, they may be present on the early earth and can be used as a good model for study the conditions and development of more complex structures in early evolution. In this work internal volume, stability and permeability of oleic acid vesicles have been investigated by electron paramagnetic resonance (EPR) method. For this purpose 80 mM oleic acid was hydrated with a 0.2 M bicine buffer (pH 8.5), containing 0.01 M spin probe. After pH adjustment with NaOH the dispersion was stirred overnight on the magnetic stirrer. As a spin probe 4-(N, N-Dimethyl-N-(2-hydroxyethyl))ammonium-2, 2, 6, 6-tetramethylpiperidine-1-oxyl (tempo-choline, ASL) was used, as due to its charge it cannot penetrate the vesicular membrane easily. LUVs were obtained by extrusion through the 100 nm polycarbonate membrane filter. To eliminate the EPR signal of non-entrapped spin probe, 20 μ l of 0.1 M sodium ascorbate, which reduces the paramagnetic nitroxide group of the spin probe to EPR non-visible hydroxylamine, was added to 20 μ l of vesicular dispersion. The captured volume Vi was determined from the EPR spectra intensity ratio of the spectra immediately after addition of ascorbic acid (I) and before addition of ascorbic acid (I0). As the intensity is proportional to the number of paramagnetic centres in the sample the ratio I/I0 is proportional to Vi/V0, where V0 is the total volume of the sample. Measuring the EPR spectra intensity decay with time after addition of ascorbate the permeability of vesicle membrane was determined and was compared to the permeability of LUV prepared from 80 mM Soya lecithin, which are better known and characterized. Stability of vesicles was measured by measuring the size of vesicles, their zeta potential by dynamic light scattering and internal volume by EPR with time after preparation of the sample.

vesicles; fatty acid; EPR

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