Anion Formation of 4‘-(Dimethylamino)-3-hydroxyflavone in Phosphatidylglycerol Vesicles Induced by HEPES Buffer: A Steady-State and Time-Resolved Fluorescence Investigation (original) (raw)
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FEBS Letters, 2001
We report on dramatic differences in fluorescence spectra of 4P P-dimethylamino-3-hydroxyflavone (probe F) studied in phospholipid membranes of different charge (phosphatidyl glycerol, phosphatidylcholine (PC), their mixture and the mixture of PC with a cationic lipid). The effect consists in variations of relative intensities at two well-separated band maxima at 520 and 570 nm belonging to normal (N*) and tautomer (T*) excited states of flavone chromophore. Based on these studies we propose a new approach to measure electrostatic potential at the surface layer of phospholipid membranes, which is based on potential-dependent changes of bilayer hydration and involves very sensitive and convenient ratiometric measurements in fluorescence emission. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
Excited State Proton Transfer and Solvent Relaxation of a 3-Hydroxyflavone Probe in Lipid Bilayers
The Journal of Physical Chemistry B, 2008
The photophysics of a ratiometric fluorescent probe, N-[[4′-N,N-diethylamino-3-hydroxy-6-flavonyl]methyl]-N-methyl-N-(3-sulfopropyl)-1-dodecanaminium, inner salt (F2N12S), incorporated into phospholipid unilamellar vesicles is presented. The reconstructed time-resolved emission spectra (TRES) unravels a unique feature in the photophysics of this probe. TRES exhibit signatures of both an excited-state intramolecular proton transfer (ESIPT) and a dynamic Stokes shift associated with solvent relaxation in the lipid bilayer. The ESIPT is fast, being characterized by a risetime of ∼30-40 ps that provides an equilibrium to be established between the excited normal (N*) and the ESIPT tautomer (T*) on a time scale of 100 ps. On the other hand, the solvent relaxation displays a bimodal decay kinetics with an average relaxation time of ∼1 ns. The observed slow solvent relaxation dynamics likely embodies a response of nonspecific dipolar solvation coupled with formation of probe-water H-bonds as well as the relocation of the fluorophore in the lipid bilayer. Taking into account that ESIPT and solvent relaxation are governed by different physicochemical properties of the probe microenvironment, the present study provides a physical background for the multiparametric sensing of lipid bilayers using ESIPT based probes.
Excited state proton transfer fluorescence of 3-hydroxyflavone in model membranes
Spectrochimica Acta Part A-molecular and Biomolecular Spectroscopy, 1997
3-Hydroxyflavone (3HF), the basic structural moiety of an important group of naturally occurring, biologically active flavonoids, has attracted extensive attention for its intramolecular excited-state proton-transfer (ESPT) and dual fluorescence characteristics. We report here, for the first time, its fluorescence (emission, excitation, polarization anisotropy and lifetime) behaviour in model membranes consisting of small, unilamellar liposomes of synthetic phosphatidylcholine (dimyristoyl phosphatidylcholine (DMPC) and dipalmitoyl phosphatidylcholine (DPPC)). The emission spectrum is conspicuously dominated by the ESPT tautomer fluorescence band. This result, along with other relevant data, indicates that the molecules are embedded in the lipid bilayer matrix facing environments where external H-bonding perturbation effects are minimized. The anisotropy (Y) versus temperature (r) profiles dramatically reveal the thermotropic gel-liquid crystalline phase transition properties of the phospholipids. signifying a novel application of the intrinsic (tautomer) fluorescence of 3HF. 0 1997 Elsevier Science B.V.
2002
The newly synthesized 3-hydroxyflavone derivative [2-(4Ј-N,N-diethylaminophenyl)-3-hydroxy-6chromonyl](N,N-dimethyl-octyl) ammonium bromide (F2) together with already used 4Ј-dimethylamino-3-HF (F) are found to be extremely sensitive to the effects of preferential hydration in model solvent system. This property is explored in the study of phospholipid vesicles of different composition made of neutral, cationic, and anionic lipids. We observe an extremely high level of response of both F and F2 fluorescence emission spectra to the surface charge of the vesicles: the N* form is strongly favored with less positively charged and more negatively charged membrane surface. The strong red-edge effects, which are almost independent of the lipid composition demonstrate the immobility of the probe environment on the time scale of fluorescence emission and suggest the static nature of hydration effects.
Flavonols - new fluorescent membrane probes for studying the interdigitation of lipid bilayers
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1998
Two flavonols, 3-hydroxy-4'-dimethylaminoflavone (FME) and 3-hydroxy-4'-(15-azacrown-5) flavone (FRC) have been investigated as new fluorescence probes for studying the formation of the interdigitated gel phase in lipid bilayers. The formation of the interdigitated gel phase in the saturated symmetrical phosphatidylcholines (PCs) and phosphatidylethanol (Peth) in the presence of ethanol has been well studied. The present study examines the behavior of these new probes in PC-ethanol and Peth-ethanol systems, as well as in PC-cholesterol and Peth-cholesterol vesicles. The present results demonstrate that both flavonols give distinctively different spectra in interdigitated lipids compared to non-interdigitated lipids, when examined in lipids in which the interdigitation behavior is known. This makes them useful for determinations of the structural state of unknown lipids, and for following the transitions between interdigitated and non-interdigitated phases. However, in the presence of cholesterol, only FCR gave appropriate indications of interdigitation. The results with FME in the presence of cholesterol were not consistent with the known behavior of the lipids examined; instead, FME appears to be located preferentially in the cholesterol-rich non-interdigitated regions of the bilayer.
Bimodal Distribution and Fluorescence Response of Environment-Sensitive Probes in Lipid Bilayers
Biophysical Journal, 2004
A remarkable heterogeneity is often observed in the spectroscopic properties of environment-sensitive fluorescence probes in phospholipid bilayers. To explain its origin, we provided a detailed investigation of the fluorescence excitation and emission spectra of 4#-dimethylamino-3-hydroxyflavone (probe F) in bilayer vesicles with the variations of fatty acid composition, polar heads, temperature, and cholesterol content. Probe F, due to excited-state intramolecular proton transfer, exhibits two bands in emission that are differently sensitive to intermolecular interactions-thereby allowing us to distinguish universal (dipole-dipole) and specific (H-bonding) interactions within the bilayer. Spectroscopic, quenching, and anisotropy data suggest the presence of two forms of probe F at different locations in the bilayer: an H-bond free form located below sn 1 -carbonyls and an H-bonded form located at the polar membrane interface. We provide a quantitative analysis of the distribution of the probe between these two locations as well as the polarity of these locations, and show that both the distribution and the polarity contribute to the probe response. Moreover, analysis of literature data on other environment-sensitive probes (Prodan, Laurdan, Nile Red, NBD lipids, etc.) in lipid bilayers allows us to suggest that the bimodal distribution in the lipid bilayer is probably a general feature of low-polar molecules with polar groups capable of H-bonding interactions.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 1997
The interaction of quinine with dimyristoylphosphatidic acid (DMPA) and dimyristoylphosphatidyl glycerol (DMPG) small unilamellar vesicles in the gel phase was studied by steady-state fluorescence spectroscopy at pHs 7. 6, 5 and 4 and 20°C. In aqueous solution, with excitation at 335 nm, the emission fluorescence spectrum of quinine varied with pH reflecting the occurrence of different charged species of the drug. In all cases, the emission maximum centered at 383 or 443 nm shifted to lower wavelength in the presence of vesicles. This indicates that the membrane-bound state quinine is in an environment of low polarity. Drug monocationic species were deeply buried in DMPG relative to DMPA bilayers whereas no significant differences were observed for dicationic species, the fluorophore being Iocated in this case in a more aqueous-like environment. Experimental association isotherms generated from fluorescence intensity changes were quantitatively analyzed in terms of the binding equilibrium model. Although the binding affinity of quinine to anionic membranes was always higher for DMPG over DMPA, dicationic species showed a reduced ability to bind the negatively charged membrane. In addition, the binding model has been related with the partition model leading to a good agreement between the theoretical (calculated from the binding model) and the experimental (from the initial slope of the experimental isotherms) partition coefficient derived in each case. 0 1997 Elsevier Science B.V.
European Biophysics Journal, 1988
Phosphoinositide metabolism in the plasma membrane is linked to transmembrane signal transduction. In this study we have investigated some physical properties (e.g. molecular order and dynamics) of phosphatidylinositol (PI) in various membrane preparations by time-resolved fluorescence techniques, using a synthetic PI derivate with a cis-parinaroyl chain on the sn-2 position. Phospholipid vesicles, normal and denervated rat skeletal muscle sarcolemmal membranes, and acetylcholine receptor rich membranes from Torpedo marmorata were investigated both at 4 °C and 20 °C. For comparison we have also included 2-parinaroyl-phosphatidylcholine (PC) in this study. The fluorescent lipids were incorporated into the membrane preparations by way of specific phospholipid transfer proteins, to ensure an efficient and non-perturbing insertion of the lipid-probes. In the Torpedo membranes the order parameters measured for the parinaroyl derivatives of both PC and PI were higher than in phospholipid vesicles. For the Torpedo membrane preparations the acyl chain order for the PI was lower than that for PC, whereas the opposite was true for the vesicles. This inversion strongly suggests that PI has different interactions with certain membrane components as compared to PC. This is also suggested by the significantly higher rate of restricted rotation of PI as compared to PC. In contrast to the order parameters, the correlation times were almost identical for both probes and showed little difference between vesicles and the Torpedo membranes. In * To whom offprint requests should be sent Iir parallel fluorescence intensity component: I± perpendicular fluorescence intensity component; SET-buffer, 0.25 M Sucrose, I mM EDTA, 10mM Tris-HC1, pH 7.4
Photochem Photobiol, 2007
3-Hydroxyflavone (3HF), a molecule that exhibits excited-state intramolecular proton transfer, has been studied for its fluorescence characteristics in dimyristoylphosphatidylcholine (DMPC) liposome membrane. 3HF partitions to the lipid bilayer membrane with a reasonably large partition coefficient. On excitation at 417 nm, a weak emission from the ground-state anion species was observed at 483 nm, whereas excitation at absorption maxima (345 nm) gives the usual intense fluorescence of the phototautomeric emission at 530 nm. In this article, we report the observation of a ground-state proton transfer reaction of 3HF in DMPC liposome membrane.