Self-quenching of nitrobenzoxadiazole labeled phospholipids in lipid membranes

The emission intensity, wavelength, and lifetime of the fluorophore nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) are sensitive to the local environmental structure when this species is present as a component of an amphiphilic membrane. Alterations of the physical and electrostatic structure of a membrane can result in changes in the fluorescence signal owing to changes in the extent of self-quenching of the probe. To investigate self-quenching, NBD-PE was incorporated into monolayers and vesicles composed of Egg phosphatidylcholine at concentrations of 0.1 to 50 mol %. Monolayer samples were dipcast onto glass slides at a pressure of 35 mN m−1. Both the integrated intensity per fluorophore (quantum yield) from vesicles and dipcast monolayers, and the mean fluorescence lifetime from vesicles decreased as the concentration of fluorophore in the membranes was increased. At all concentrations 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 did not permit the use of standard theoretical models such as the Klafter–Blumen or Stern–Volmer equations which are normally employed to describe changes in fluorescence lifetime with changes in quencher concentration. Instead, a phenomenological approach was used to develop an empirical model of fluorescence self-quenching which could describe the observed alterations in the fluorescence lifetime and intensity. The model was based on a combination of Perrin quenching and Förster energy transfer. The fluorescence data was fit by a model wherein NBD-PE formed nonemissive trap sites with a critical radius of Rc=1.0±0.1 nm (Perrin quenching), with Förster energy transfer occurring to the trap sites with an R0 value of 2.55±0.10 nm as determined from spectral overlap integrals.