Chlorinated hydrocarbon-cell membrane interactions studied by the fluorescence quenching of carbazole-labeled phospholipids: probe synthesis and characterization of the quenching methodology.

In recognition of the need to understand better the interactions of the chlorinated hydrocarbon insecticides with cell membranes we investigated the use of fluorescence quenching of membrane-bound fluorophores by these chlorinated hydrocarbons. An extensive survey of potential fluorophores identified the N-alkyl derivatives of carbazole as being especially suitable fluorophores. The fluorescence emission of these derivatives is quenched by a wide variety of commonly-used chlorinated hydrocarbons. This quenching is collisional and does not result in significant photodecomposition. Four structurally distinct carbazole-labeled phospholipids were synthesized, and their structures were confirmed by 270 MHz proton NMR and by chromatographic and chemical means. The carbazole moiety of each labeled phospholipid should be localized at a different depth in lipid bilayer. However, water soluble quenchers indicate that the fluorophores are inaccessible to the aqueous phase, irrespective of their point of attachment to the phospholipids. When incorporated into lipid bilayers, the fluorescence lifetime of these carbazole-labeled phospholipids reveals the collisional frequency between the fluorophore and the chlorinated hydrocarbon. As a result quenching of membrane-bound fluorophores may be used to measure: (1) the diffusional rate of the chlorinated hydrocarbon in the bilayer; (2) the lipid-water partition coefficient; (3) the maximum binding capacity of the membrane for the chlorinated hydrocarbon. Examples of all these measurements are given, and the fluorometric results are confirmed by direct chemical analysis.