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.