FRET between non-substrate probes detects lateral lipid domain formation during phospholipase A2 interfacial catalysis.

The probes C(12)-NBD-FA (12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)] dodecanoic acid) and C(18)-R (octadecyl rhodamine B chloride) have been used as donor and acceptor, respectively, in FRET studies on liposomes subjected to pancreatic PLA(2) action. Neither of these fluorophores is a substrate for the enzyme but one of them, C(12)-NBD-FA, is an analog of the fatty acid reaction product. The fluorophores were incorporated into 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes and FRET was studied following the fluorescence of the donor, C(12)-NBD-FA. Working with a molar ratio of acceptor to donor (A/D) of 1, we have found that FRET efficiency (E) decreases during DPPC hydrolysis. After 60min, the decrease is equivalent to a reduction of more than five times in the effective A/D ratio, as estimated by interpolation in an efficiency vs. A/D reference curve. Using a more complete, empirical approach, the efficiency data, calculated from experiments at variable A/D proportions and constant donor concentration, were fitted by a rectangular hyperbolic function. The parameter K of this function, representing the A/D ratio at half-maximum transfer efficiency, increases more that five times after 60min hydrolysis. This agrees with the reduction of the effective acceptor density sensed by the donor after hydrolysis, detected by the interpolation procedure. The heterogeneous distribution of acceptor and donor induced by hydrolysis can be attributed to the formation of product domains in the phospholipid membranes and is consistent with the preferential segregation of the donor, which is an analog of the fatty acid reaction product, in those domains. In conclusion, FRET between non-substrate probes detects the heterogeneities generated in phospholipid membranes by PLA(2) action.

Lateral organization of mixed, two-phosphatidylcholine liposomes as investigated by GPS, the slope of Laurdan generalized polarization spectra.

The effect of the excitation or emission wavelengths on Laurdan generalized polarization (GP) can be evaluated by GPS, a quantitative, simplified determination of the GP spectrum slope, the thermotropic dependence of which allows the assessment of phospholipid lamellar membrane phase, as shown in a recent publication of our laboratory [J.B. Velázquez, M.S. Fernández, Arch. Biochem. Biophys. 455 (2006) 163-174]. In the present work, we applied Laurdan GPS to phase transition studies of mixed, two-phosphatidylcholine liposomes prepared from variable proportions of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). We have found that the GPS function reports a clear limit between the gel/liquid-crystalline phase coexistence region and the liquid-crystalline state, not only at a certain temperature T(c) for liposomes of constant composition submitted to temperature scans, but also at a defined mole fraction X(c), for two-component liposomes of variable composition at constant temperature. The T(c) or the X(c) values obtained from GPS vs. temperature or GPS vs. composition plots, respectively, allow the construction of a partial phase diagram for the DMPC-DPPC mixtures, showing the boundary between the two-phase coexisting region and the liquid-crystalline state. Likewise, at the onset of the transition region, i.e., the two-phase coexisting region as detected by GPS, it is possible to determine, although with less precision, a temperature T(o) or a mole fraction X(o) defining a boundary located below but near the limit between the gel and ripple phase, reported in the literature. These GPS results are consistent with the proposal by several authors that a fraction of L(alpha) phospholipids coexists with gel phospholipids in the rippled phase.