Organization and dynamics of pyrene and pyrene lipids in intact lipid bilayers. Photo-induced charge transfer processes

Abstract

The dynamics of fluorescence quenching and the organization of a series of pyrene derivatives anchored in various depths in bilayers of phosphatidylcholine small unilamellar vesicles was studied and compared with their behavior in homogeneous solvent systems. The studies include characterization of the environmental polarity of the pyrene fluorophore based on its vibronic peaks, as well as the interaction with three collisional quenchers: the two membrane-soluble quenchers, diethylaniline and bromobenzene, and the water soluble quencher potassium iodide. The system of diethylaniline-pyrene derivatives in the membrane of phosphatidylcholine vesicles was characterized in detail. The diethylaniline partition coefficient between the lipid bilayers and the buffer is approximately 5,800. Up to a diethylaniline/phospholipid mole ratio of 1:3 the perturbation to membrane structure is minimal so that all photophysical studies were performed below this mole ratio. The quenching reaction, in all cases, was shown to take place in the lipid bilayer interior and the relative quenching efficiencies of the various probe molecules was used to provide information on the distribution of both fluorescent probes and quencher molecules in the lipid bilayer. The quenching efficiency by diethylaniline in the lipid bilayer was found to be essentially independent on the length of the methylene chain of the pyrene moiety. These findings suggest that the quenching process, being a diffusion controlled reaction, is determined by the mobility of the diethylaniline quencher (with an effective diffusion coefficient D approximately 10(-7) cm2 s-1) which appears to be homogeneously distributed throughout the lipid bilayer. The pulsed laser photolysis products of the charge-transfer quenching reaction were examined. No exciplex (excited-complex) formation was observed and the yield of the separated radical ions was shown to be tenfold smaller than in homogenous polar solutions. The decay of the radical ions is considerably faster than the corresponding process in homogenous solutions. Relatively high intersystem crossing yields are observed. The results are explained on the basis of the intrinsic properties of a lipid bilayer, primarily, its rigid spatial organization. It is suggested that such properties favor ion-pair formation over exciplex generation. They also enhance primary geminate recombination of initially formed (solvent-shared) ion pairs. Triplet states are generated via secondary geminate recombination of ion pairs in the membrane interior. The results bear on the general mechanism of electron transfer processes in biomembranes.

Lateral Organization of Pyrene-labeled Lipids in Bilayers as Determined from the Deviation from Equilibrium between Pyrene Monomers and Excimers

“…Our previous study (18) indicates that the aromatic amine DEA, which quenches excited pyrene moieties through a charge transfer mechanism, is a more efficient quencher (k q in ethanol at 25°C is 1 ϫ 10 10 M Ϫ1 s…”

“…2. All (except the highest) DEA concentrations used were below the DEA/lipid concentration, which may have a large effect on bilayer properties (18). For pyrene (a) and pyrene valerate (b) the slopes of the DEA Stern-Volmer plots for M* and E are identical (K E ϭ K M ), which is not the case for pyrene decanoate (c) and pyrene hexadecanoate (d), for which K M Ͼ K E .…”

“…This was not studied in detail, although such nonlinearity is consistent with the presence of two populations of fluorophores, one of which is not accessible to the quencher. This may be an artifact, as the highest concentration of DEA used (DEA/lipid ratio Ͼ 0.4) may have a large effect on membrane structure (18). The two different criteria represented by Figs.…”

“…18 and references therein). The most common use of pyrene and pyrene lipid is based on the ability of one excited pyrene moiety, together with a nonexcited pyrene moiety, to form an excited-state dimer (excimer).…”

“…One of the advantages of DEA as a quencher is its high membrane-tobuffer partition coefficient (18). In order to study which one of the excited species of pyrene (monomer or excimer) is quenched by DEA, we characterized the quenching of pyrene in ethanol under two sets of conditions: (a) pyrene at 3.6 ϫ 10 Ϫ6 M, at which no excimers can be detected and (b) pyrene at 1.8 ϫ 10 Ϫ2 M, at which most of the fluorescence intensity is associated with the excimer peak at 480 nm (E/M ϭ 6).…”