SELF-QUENCHING OF NITROBENZOXADIAZOLE LABELED PHOSPHOLIPIDS IN LIPID-MEMBRANES

The emission intensity, wavelength, and lifetime of the fluorophore ni trobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) are sen sitive to the local environmental structure when this species is prese nt as a component of an amphiphilic membrane. Alterations of the physi cal and electrostatic structure of a membrane can result in changes in the fluorescence signal owing to changes in the extent of self-quench ing of the probe. To investigate self-quenching, NBD-PE was incorporat ed into monolayers and vesicles composed of Egg phosphatidylcholine at concentrations of 0.1 to 50 mol %. Monolayer samples were dipcast ont o glass slides at a pressure of 35 mN m-1. Both the integrated intensi ty per fluorophore (quantum yield) from vesicles and dipcast monolayer s, and the mean fluorescence lifetime from vesicles decreased as the c oncentration of fluorophore in the membranes was increased. At all con centrations studied the decay of NBD-PE fluorescence was fitted to two discrete exponentials, and both lifetime components were observed to change with concentration. The complexity of the fluorescence decay di d not permit the use of standard theoretical models such as the Klafte r-Blumen or Stern-Volmer equations which are normally employed to desc ribe changes in fluorescence lifetime with changes in quencher concent ration. Instead, a phenomenological approach was used to develop an em pirical model of fluorescence self-quenching which could describe the observed alterations in the fluorescence lifetime and intensity. The m odel was based on a combination of Perrin quenching and Forster energy transfer. The fluorescence data was fit by a model wherein NBD-PE for med nonemissive trap sites with a critical radius of R(c) = 1.0 +/- 0. 1 nm (Perrin quenching), with Forster energy transfer occurring to the trap sites with an R(0) value of 2.55 +/- 0.10 nm as determined from spectral overlap integrals.