Study of Heterogeneous Emission of Parinaric Acid Isomers Using Multifrequency Phase Fluorometryt

The fluorescence lifetimes of parinaric acid (PnA) isomers have been measured in pure solvents and in synthetic phospholipid membranes over a wide temperature range. In pure solvents, dodecane, ethanol, and cyclohexanol, the emission is well described by three exponential components of approximately 40, 12, and 2 ns. The preexponential factor of the long-lifetime component is quite small, and this component is neglected in the present study. The preexponential factors of the shorter lifetime components are strongly temperature dependent. Both cisand trans-PnA show an inversion of the fractional contribution of these two components in a narrow temperature range. The inversion temperature is lower for trans-PnA than for cis-PnA, both in ethanol and D u r i n g the past decade, fluorescence methodologies have been among the most widely utilized approaches to characterizing the physical state of membranes. In particular, fluorescent probes which partition preferentially into membranes have been extensively utilized to monitor lipid fluidity (Shinitzky & Barenholz, 1974). The naturally fluorescent fatty acids cis,trans,trans,cis9,11,13,15-octadecatetraenoic acid (cis-PnA)' and trans-PnA seemed particularly appropriate for such studies because of their high quantum yields in membranes (relative to the aqueous environment) and their structural analogies to intrinsic membrane components which reduce uncertainties about thier position in the bilayer and render them less perturbing than other more bulky probes. A different partition coefficient between the gel and liquid-crystal phases of the bilayer has been reported for the two isomers of PnA; these results were based on studies of the decay rates and polarization values of PnA fluorescence in pure solvents (Sklar et al., 1977a) and in synthetic phospholipid membranes (Sklar et al., 1975, 1977b). Specifically, transPnA is reported to partition preferentially into the gel phase while cis-PnA reportedly does not favor one phase over the other. Moreover, in pure solid phospholipids, both PnA isomers are reported to be characterized by a double exponential decay, with a low concentration of short-lived fluorophores, while in pure fluid phospholipids the probes are reported to give a double exponential decay but with a low concentration of long-lived fluorophores. These results have been explained by the persistence of some degree of disorder in the gel phase and some degree of order in the liquid-crystal phase, which are detectable by the PnA isomers. Specifically, the short-lived and long-lived components correspond to fluorophores present in fluid and solid phases, respectively. The prerequisite for such an interpretation is the monoexponential decay of the PnA From the Department of Chemistry, University of Rome, Rome 00187, Italy (T.P. and F.C.), and the Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (E.G.). Receiued March 19,1984. This research was supported by Grant ICRPhysics 1-2-22290 from the Department of Physics, University of Illinois at Urbana-Champaign. 0006-2960/84/0423-5660$0 1.50/0 in dodecane. The different temperature behavior of the decay of the two PnA isomers can influence their behavior in phospholipids. In L-a-dimyristoylphosphatidylcholine (DMPC) and L-a-dipalmitoylphosphatidylcholine (DPPC) membranes, the emission is more complex, and more than three components are necessary to describe the fluorescence decay accurately. On the basis of their different behavior in pure solvents, we anticipate that cis-PnA will detect the phospholipid transition in DMPC and DPPC at lower temperature. In fact, this effect has been observed, but it has been attributed to different partitioning of the two isomers in the phospholipid phases rather than to their different photophysics as we suggest here. isomers in pure solvents. Such results have been recently reported in a pulsed lifetime study utiziling synchrotron radiation (Wolber & Hudson, 1981). The aim of the present study was to verify the decay modes for PnA isomers in pure isotropic solvents and in synthetic phospholipid bilayers to critically evaluate the hypothesis that the different lifetime components can arise from different phases coexisting in model and natural membranes. For our studies, we utilized the powerful new fluorescence methodology of multifrequency cross-correlation phase and modulation fluorometry (Jameson et al., 1984). Using the instrumentation described by Gratton & Limkeman (1983) which allows facile selection of arbitrary light modulation frequencies, we were able to obtained lifetime results with resolutions of several picoseconds. Such multifrequency results permit the resolution of heterogeneous emission into the component lifetimes and the relative fractional weights. Our results demonstrate that the decay of both PnA isomers in the three pure isotropic solvents utilized is best characterized by three exponentials with relative amplitudes which are highly temperature dependent. These results render questionable the previous partition coefficient determinations for the two PnA isomers in phospholipid bilayers (Sklar et al., 1977b). In addition, the results for the two PnA's in pure phospholipids were more complex than triple exponential decays. Materials and Methods cis-PnA and trans-PnA were purchased from Molecular Probes Co. The contents of the vials were solubilized in pure ethanol with 0.1% BHT as antioxidant. Prior to use, the PnA-ethanol solution was passed through a silica cartridge (Sep-Pak, Waters Associates, Inc.) and washed once with ethanol in the presence of 0.1% BHT. With this treatment, contaminants and degradation products are reported to remain Abbreviations: cis-PnA, cis,trans,trans,cis-9,11,13,15-octadecatetraenoic acid; trans-PnA, all-trans-9,11,13,15-octadecatetraenoic acid; BHT, butylated hydroxytoluene; DMPC, L-a-dimyristoylphosphatidylcholine; DPPC, r-a-dipalmitoylphosphatidylcholine; HPLC, high-pressure liquid chromatography; PBS, phosphate-buffered saline; DPH, 1,6-diphenyl-l,3,5-hexatriene.