Conjugated polyene fatty acids as fluorescent membrane probes: model system studies.
The use of conjugated polyene fatty acids as probes of membrane structure is examined, alpha- and beta-parinaric acid (cis, trans, trans, cis- and all trans-9, 11, 13, 15-octadecatetraenoic acid) and synthetic lecithins containing an alpha-parinaric acid chai in position 2 have been prepared, and their absorption and fluorescence properties have been determined. Their absorption spectra are at sufficiently long wavelength to be unobscured by cellular chromophores such as nucleotides and aromatic amin acids. Parinaric acid absorption does, however, overlap tryptophan emission which allows fluorescence energy transfer. Potential uses of these fluorescent probes are presented with studies on mode systems with known physical properties. Dipalmitoyl phosphatidylcholine exhibits a sharp phase transition 1 degree wide at 42 degrees C, as monitored by the fluorescence intensit of parinaric acid. The magnitude of the transition is independent of probe concentration, but the width of the transition and hysteresis are dependent upon such factors as the probe concentration and whether or not sonication is used in sample preparation. Using both fluorescence and absorption properties of the probe, we show that the addition of cholesterol to the dispersion broadens and decreases the magnitude of the transition. These results are interpreted in terms of a change in the polarizability of the acyl chains of a lipid bilayer undergoes a thermal transition. Lipid-protein interactions are studied by the binding of alpha-parinaric acid to bovine serum albumin. Fluorescence enhancement, absorption spectral shifts, and quenching of tryptophan fluorescnece are observed when alpha-parinaric acid binds to bovine serum albumin. Calculations based on these measurements are consistent with two binding sites of KB approximately 10(8) (M-1) and three to four binding sites of KB approximately 10(6)-10(7) (M-1), similar to known values for the binding of other long-chain fatty acids. Biosynthetic incorporation of beta-parinaric acid into the E. coli fatty acid auxotroph 30E betaox has been accomplished and phase transitions in cells and isolated phospholipids are shown.
D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol; D004926:Escherichia coli / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated; D008433:Mathematics; D008567:Membranes, Artificial; D008954:Models, Biological; D010713:Phosphatidylcholines; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics
D005231:Fatty Acids, Unsaturated; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D002784:Cholesterol