Fluorescence quenching of fluorescein by Merocyanine 540 in liposomes (original) (raw)
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Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes
Biophysical Journal, 1989
In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membranes to explain the fluorescence quenching of 1-palmitoyl-2-(10-Ipyren-1-yl]decanoyl)-sn-glysero-3-phosphatidylcholine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 108 M-'s-1 for the pyrene monomer quenching and 1.3 x 107 M-'s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10-7 cm2s-' for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.
Journal of Biomedical Optics, 2008
The time-resolved fluorescence of photosensitizers ͑PSs͒ of varying hydrophobicities, di-and tetrasulfonated Al phthalocyanines ͑Al-2 and Al-4͒, and Photochlor® ͑HPPH͒, was investigated in liposomes used as cell-mimetic models. Using frequency-and timedomain apparatus, the fluorescence lifetime, fluo , was compared for PSs free in aqueous solution and in a liposome-associated state at varied temperatures ͑25 to 78°C͒ and oxygen concentrations ͑0-190 M͒. The analysis of fluo revealed different decay behaviors for the free-solution and liposome-confined PSs, most significantly for the lipophilic HPPH. Hydrophilic PS drugs ͑Al-4, Al-2͒ were less affected by the liposomal confinement, depending on the relative hydrophilicity of the compound and the consequent localization in lipsomes. Changes in the emission decay due to confinement were detected as differences in the lifetime between the bulk solution and the liposome-localized PS in response to heating and deoxygenation. Specifically, hydrophilic Al-4 produced an identical lifetime trend as a function of temperature both in solu and in a liposome-confined state. Hydrophobic HPPH exhibited a fundamental transformation in its fluorescence decay kinetics, transitioning from a multiexponential ͑in free solution͒ to single-exponential ͑in liposome͒ decay. Deoxygenation resulted in a ubiquitous fluo increase for all PSs in free solution, while the opposite, a fluo decrease, occurred in all liposomal PSs.
A Stability Test of Liposome Preparations Using Steady-State Fluorescent Measurements
Drug Delivery, 2001
The stability of liposome preparations under the action of the nonionic detergent Triton X-100 was measured using the fluorescent molecular probe octadecylrhodamine B (R18). The probe inserted in the lipid bilayer shows a self-quenched fluorescence and the degree of quenching depends both on the probe concentration and the phase state of the lipid membrane. The addition of detergent to the liposomes produces a steep decrease in self-quenching caused by dilution of the probe in the bilayer. The curves of steady-state fluorescence intensity show an abrupt change in slope that corresponds to the point at which liposomes break down into lipid-detergent mixed entities that are different from the earlier liposome-monodisperse population. The lytic process was followed in parallel by dynamic light scattering (DLS), and the analysis of the DLS results agree with the interpretation of the fluorescence measurements. The probe R18 therefore is a useful marker to test the stability of liposome ...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
The structural dynamics of the main phase transition of large unilamellar dinervonoylphosphocholine (DNPC) vesicles was investigated by steady state and time-resolved fluorescence spectroscopy of the membrane incorporated fluorescent lipid analog, 1-palmitoyl-2[10-(pyren-1yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC). These data were supplemented by differential scanning calorimetry (DSC) and fluorescence anisotropy measured for 1-palmitoyl-2-(3-(diphenylhexatrienyl) propanoyl)-sn-glycero-3-phosphocholine (DPHPC). The collected data displayed several discontinuities in the course of the main transition and the pretransition. The discontinuities seen in the fluorescence properties may require modification of the existing models for phospholipid main transition as a first order process. From our previous study on dipalmitoylphosphocholine (DPPC), we concluded the transition to involve a first-order process resulting in the formation of an intermediate phase, which then converts into the liquid crystalline state by a second order process. Changes in the physical properties of the DNPC matrix influencing probe behavior were similar to those reported previously for PPDPC in DPPC. In gel state DNPC [(T À T m ) < À 10] the high values for excimer/monomer emission ratio (I e /I m ) suggest enrichment of the probe in clusters. In this temperature range, excimer fluorescence for PPDPC (mole fraction X PPDPC = 0.02) is described by two formation times up to (T À T m ) c À 10, with a gradual disappearance of the fractional intensity (I R1 ) of the shorter formation time (s R1 ) with increasing temperature up to (T-T m ) c À 10. This would be consistent with the initiation of the bilayer melting at the PPDPC clusters and the subsequent dispersion of the one population of PPDPC domains. A pronounced decrement in I e starts at (T-T m ) = À 10, continuing until T m is reached. No decrease was observed in fluorescence quantum yield in contrast to our previous study on DPPC/PPDPC large unilamellar vesicles (LUVs) [J. Phys. Chem., B 107 (2003) 1251], suggesting that a lack of proper hydrophobic mismatch may prevent the formation of the previously reported PPDPC superlattice. With further increase in temperature and starting at (T À T m ) c À 1, I e , s R2 , and excimer decay times (s D ) reach plateaus while increment in trans ! gauche isomerization continues. This behavior is in keeping with an intermediate phase existing in the temperature range À 1 < (T À T m ) < 4 and transforming into the liquid disordered phase as a second order process, the latter being completed when (T À T m ) ! 4 and corresponding to c 50% of the total transition enthalpy. D change; I e /I m , ratio of pyrene excimer and monomer fluorescence intensity; Int I e , integrated excimer intensity of the time-resolved fluorescence emission; LUV, large unilamellar vesicle; MLV, multilamellar vesicle; PPDPC, 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine; T, temperature; T m , main phase transition temperature; T p , pretransition temperature; X lipid , mole fraction of the indicated lipid; s R , rise time (excimer formation time); s D , excimer decay time; s M , weighted average monomer lifetime
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1987
Five lipophilic 21-peptide analogs of the potential-dependent pore-former, alamethicin, were synthesized bearing tryptophan residues at the position 1, 6, 11, 16 and 21 on a long, conformationally rigid, a-helix. The a-helical conformation was induced and stabilized using the sequential oligomers (Ala-Aib-Ala-Aib.Ala)n as analyzed by CI) and NMR. The partitioning of the N-t-butoxycarbonyi 21-peptide methyl esters and the N-terminally deprotected a-helices was followed by fluorescence enhancement in phospholipid bilayer vesicles. Quenching experiments were performed by titrating with n-doxyl stearic acids bearing the nitroxide label at positions 5, 7, 10, 12 and 16. This well-defined system revealed that the N-and C-terminal tryptophan residues become situated in the hydrophilic region. Tryptophan at position 11 was found in the lipophilic core, whereas the tryptophan at positions 6 and 16 were localized at intermediate depths of the lipid membrane. Therefore, the helices span the lipid bilayer with their long axis normal to the membrane surface.
Archives of Biochemistry and Biophysics, 1996
gested to have a chemical structure in which the angle between the absorption and emission dipole moments Motional properties of fluorescent substances prois very large. On the basis of these observations, the duced by lipid peroxidation by a time-resolved fluoproduction pathway of fluorophores in oxidized memrescence polarization technique were studied. When branes is discussed. ᭧ 1996 Academic Press, Inc. liposomes containing phosphatidylethanolamine (PE) Key Words: amino phospholipid; anisotropy; fluoand linoleic hydrocarbon chain were incubated at rescence; liposome, peroxidation. 37ЊC, fluorophores absorbing maximally at 360 nm and emitting near 430 nm were produced. Their fluorescence anisotropy decay measured at 23ЊC was fitted well with a sum of a fast relaxation and a time-inde-Lipid peroxidation alters several physical properties pendent residual term. With the increase of oxidation of biomembranes. For example, membrane proteins are degree, the time constant of the relaxation term incrosslinked, and their rotational and translateral mocreased. This may be explained by alteration in the bility is decreased (1). In the lipid domain, lipid peroximembrane structure or by modification of the fluoresdation causes an enhancement of flip-flop movements cent products themselves. Information on the location of phospholipids (2, 3), influences polymorphic phase of the fluorescent products was obtained when their behavior of lipids (4), and alters the membrane fluidity motional property was compared with those of various (5, 6). In addition, peroxidation can inactivate enzymes extrinsic probes that were incorporated at different and cause structural abnormalities of biomembranes. positions of the lipid bilayer. It was found that the Some of the abnormalities are concomitant with formamotional property of the fluorescent oxidation prodtion of fluorescent substances, which are produced ucts is similar to that of 1-(4-trimethylammoniummostly by the reaction of lipid oxidation products with phenyl)-6-phenyl-1,3,5-hexatriene, a rod-shaped hyprimary amino compounds. This reaction has been drophobic probe with a charged terminal. Other shown to be responsible for the accumulation of fluoprobes sensing the polar region or the hydrophobic rescent pigments in aged cells (7, 8). region of the membrane were characterized by a lower So far, three types of model reactions have been proorder parameter. It is suggested that the fluorescent posed to describe production of fluorescent substances oxidation products have a polar moiety located at the by peroxidation in the presence of amino compounds: membrane surface and attached to the amino group of PE while the tail part being buried in the hydrophobic (1) malondialdehyde (MDA), 2 one of the major products region of the membrane. This picture is supported by fluorescence quenching experiments with the aqueous
Biochemistry, 1988
Addition of the quaternary ammonium detergent [ [ [ (1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl]dimethylbenzylammonium hydroxide (DEBDA [OH]) and the fluorescent probes N-(7-nitro-2,1,3-benzoxadiazol-4-y1)phosphatidylethanolamine and N-(lissamine rhodamine B sulfony1)phosphatidylethanolamine (N-NBD-PE and N-Rh-PE, respectively) to liposomes composed of phosphatidylcholine (PC) and cholesterol (chol) resulted in the formation of fluorescently labeled liposomes bearing DEBDA[OH]. Incubation of the anionic polymer poly(aspartic acid) (PASP) with such liposomes resulted in strong agglutination, indicating an association between the negatively charged PASP and the positively charged liposome-associated DEBDA[OH]. Addition of PASP to a mixture of fluorescently labeled and nonlabeled liposomes, both carrying DEBDA[OH], resulted in a significant increase in the extent of fluorescence, namely, fluorescence dequenching. The degree of the fluorescence dequenching was dependent upon the ratio between the nonfluorescent and the fluorescent liposomes, upon the temperature of incubation, and upon the amount * Author to whom correspondence should be addressed.
Biophysical Journal, 1995
A detailed photophysical study of the fluorescence quenching (transient and steady state) of the macrolide antibiotic filipin by nitroxide-substituted fatty acids and a cholesterol derivative was carried out, aimed at determining its transverse position in a model system of membranes (multilamellar vesicles of dipalmitoylphosphatidylcholine). Filipin partitions efficiently into membranes (Kp = (5.0 + 1.0).103; 200C) and it was concluded that the antibiotic is buried in the membrane, away from the lipid-water interface. In addition, information on the organization of the quenchers was also obtained. The 5-nitroxide derivative of the fatty acid is essentially randomly distributed, while the 1 6-nitroxide is aggregated at concentrations higher than-5% molar. For the cholesterol compound the results point to a phase separation at concentrations higher than 3% molar (below this limit concentration filipin associates with the derivatized sterol with KA = 20 M-1, assuming a 1:1 interaction). We propose that this phase separation and the aggregation state of filipin in the aqueous solution may be key processes in the antibiotic mode of action. A systematic and general approach to fluorescence quenching data analysis in complex (e.g., biochemical) systems is also presented.
Journal de physique, 2004
The interaction of the anionic probe Merocyanine 540 (MC540) with mono and bilayers of different lipid composition has been analyzed in order to get insight in the lipid charge influence on probe activity. The Langmuir-Blodgett technique showed that MC540 was able to incorporate predominantly into cationic lipid monolayers composed of Dimyristoyl phosphatidyl choline ! Dimyristoyl trimethyl ammonimnpropane (DMPC/DMPTAP) (75 : 25, mol : mol). However, at low surface pressures, the probe could also allocate into the interface of the zwitterionic Dimyristoyl phosphatidyl choline (DMPC) and the anionic Dimyristoyl phosphatidyl cholinel Dimyristoyl phosphatidyl glycerol (DMPC/DMPG) (75 : 25, mol mol) thus indicating a low interaction with these lipid compositions. These results were in agreement with the studies performe with Large Unilamelar Vesicles (LUVs) of the same lipid compositions based in the spectroscopic analysis of probe absorption maxima modification upon its incorporation into the lipid bilayers.
Journal of Photochemistry and Photobiology A: Chemistry, 1995
Fluorescence energy transfer in lipid vesicles between N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)-labelled phosphatidylethanolamine (acting as donor) and N-(lissamine-rhodamine B)-labelled phosphatidylethanolamine (acting as acceptor) was studied by steady state and time-resolved fluorescence quenching analysis. Both fluorescent phospholipids were incorporated as minor components in four different types of lipid vesicle: dipalmitoylphosphatidylglycerol vesicles in their L~ gel phase at 20 °C and in their La liquid crystalline phase at 50 °C, and egg yolk phosphatidylethanolamine vesicles at 40 °C in their L,, liquid crystalline phase at pH 9.5 and in their Hn inverted hexagonal phase at pH 5.0. The quenching of the donor fluorescence by energy transfer is diffusion controlled in all cases, except in the L~ gel phase. The dimensionality and type of constraints imposed on diffusion are different in each case, with the most efficient diffusion-controlled quenching in the hexagonal phase.