The response of fluorescent amines to pH gradients across liposome membranes (original) (raw)
Related papers
Intracellular pH measurements using the fluorescence of 9-aminoacridine
FEBS Letters, 1984
9-Aminoacridine is a fluorescent weak base which distributes across the plasma membrane according to the pH gradient, and can be used to monitor changes in intracellular pH in eukaryotic cells. A calibration technique using the ionophore nigericin is described which allows us to measure intracellular pH values of chick skeletal muscle cells.
The ‘ΔpH’-probe 9-aminoacridine: response time, binding behaviour and dimerization at the membrane
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988
The fluorescence quenching of 9-aminoacridiue (9-AA) after imposition of a transmembrane pH gradient (inside acidic) in liposomes has been investigated for a number of different lipid systems. The initial fluorescence decrease after a rapid pH jump, induced in the extravesicular medium by a stopped-flow mixing technique, was ascribed to a response of 9-AA to the imposed pH gradient and not to changes in the vesicular system itself. Time constants for this fluorescence quenching are in the range of several hundred milliseconds at 25 °C. Fluorescence recovery which should be correlated to the dissipation of the pH gradient occurs in the 100 s time range and is 10-30-times faster than the dpH decay monitored with the entrapped hydrophilic pH-indicator dye pyranine. The quenching was severely hindered below the lipid phase transition of dipalmitoyiphosphatidylglyceroi. No d pH-induced quenching was obtained in lipid vesicles containing only zwitterionic, net uncharged phosphatidylcholine headgroups. For the occurrence of quenching, the presence of negatively charged headgroups, i.e. phosphatidyiglyceroi or phosphatidylserine, was necessary. The extent of quenching, at a specific pH difference applied, had a cooperative dependency (Hill coefficient ~-2) on the number of negative headgroups in the membrane and on the concentration of unquenched (unbound) 9-AA molecules. The concentration of quenched 9-AA molecules was furthermore proportional to the number of dimer-excimer complexes of 9-AA which are formed during the quenching process.
Journal of Biomedical Optics, 2005
Liposomes are known to be taken up by the liver cells after intravenous injection. Among the few techniques available to follow this process in vivo are perturbed angular correlation spectroscopy, nuclear magnetic resonance spectroscopy, and scintigraphy. The study of the intracellular pathways and liposomal localization in the different liver cells requires sacrifice of the animals, cells separation, and electronic microscopy. In the acidic intracellular compartments, the in situ rate of release of liposomes remains poorly understood. We present a new method to follow the in situ and in vivo uptake of liposomes using a fluorescent pH-sensitive probe 5,6carboxyfluorescein (5,6-CF). 5,6-CF is encapsulated in liposomes at high concentration (100 mM) to quench its fluorescence. After laparotomy, liposomes are injected into the penile vein of Wistar rats. Fluorescence images of the liver and the skin are recorded during 90 min and the fluorescence intensity ratio is calculated. Ratio kinetics show different profiles depending on the liposomal formulation. The calculated intracellular liver pH values are, respectively, 4.5 to 5.0 and 6.0 to 6.5 for DSPC/chol and DMPC liposomes. After sacrifice and flush with a cold saline solution, the pH of the intracellular site of the liver (ex vivo) is found to be 4.5 to 5.0. This value can be explained by an uptake of liposomes by the liver cells and subsequent localization into the acidic compartment. An intracellular event such as dye release of a drug carrier (liposomes loaded with a fluorescent dye) can be monitored by pH fluorescence imaging and spectroscopy in vivo and in situ.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1993
Liposomes of egg PC/PG (8:2, mol/mol) were multilabelled with PBFI, pyranine and oxonol VI, fluorescent probes for, respectively, K +, H + and membrane potential. Monitoring fluorescence with a multichannel photoncounting spectrofluorometer during K + filling experiments allowed to measure K ÷ influx, the associated H ÷ efflux and the membrane potential, continuously and simultaneously. The proton net efflux quantitatively mirrored the K ÷ net influx. The rate of the K÷/H + exchange diminished progressively as a quasi-equilibrium was reached for both K ÷ and H ÷. In the presence of valinomycin, the measured membrane potential during the K + filling actually corresponded to the Nernst potential calculated from the observed K + gradient. In the absence of valinomyein, it corresponded to the Nernst potential calculated from the observed H ÷ gradient. In the latter case, the permeability coefficient of liposomes to K ÷, calculated from the Goldman-Hodgkin-Katz relation, was 6.10-13 m s-1. The selectivity sequence for alkali cations of liposomes was determined from the measured H ÷ efflux associated to the influx of the different cations. The selectivity sequence corresponded to the series VI of Eisenman, suggesting interaction of the cation with an anionic field of intermediate strength.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988
The sudace density of the fluorescent probe N-(lissamine Rbodamine B sulfonyl)dipalmitoylphosphatidylcholine is the same in the two lipid leaflets of phosphatidylcholine bilayers containing the probe. In the liquid-crystalline state, the probe molecules aggregate above a threshold amount, approximately 0.2 mol/mol phospholipids. Above this threshold value, the surface density of the free probe molecules is constant, and all probe molecules added are incorporated in the aggregated form. The aggregation of the probe increases by approximately 20% when the medium pH is lowered to 4. In the gel state, the probe aggregation is higher than tlmt in the liquid-crystalline state, and the free probe molecules distribute unevenly in the bilayer surface. Even though the results obtained in our model system cannot be directly extrapolated to all model systems, we point out that care is to be taken in the use of the probe. In fact, only in membranes in the liquid-crystalline state in which the amount of probe molecules to phospholipid molecules is lower than 1 : 7 the fluorescence response of the probe is independent of the pH changes and of the molecular aggregation.
The Journal of Membrane Biology, 1982
port have, in the main, utilized artificial membranes, both planar and vesicular. Systems of biological interest, viz., cells and organelles, resemble vesicles in size and geometry. Methods are, therefore, required to extend the results obtained with planar membranes to liposome systems. In this report we present an analysis of a fluorescence technique, using the divalent cation probe chlortetracycline, in small, unilamellar vesicles, for the study of divalent cation fluxes. An ion carrier (X537A) and a pore former (alamethicin) have been studied. The rate of rise of fluorescence signal and the transmembrane ion gradient have been related to transmembrane current and potential, respectively. A second power dependence of ion conduction-including the electrically silent portion thereof-on X537A concentration, has been observed. An exponential dependence of "current" on "transmembrane potential" in the case of alamethicin is also confirmed. Possible errors in the technique are discussed.
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
Photochemical & Photobiological Sciences, 2006
The partitioning efficiency of neutral and anionic prototropic forms of 1-hydroxypyrene in liposome suspensions has been studied. The high partition coefficient value of 1-hydroxypyrene indicates an easy incorporation of the molecule into the lipid bilayer. Detailed pH studies indicate that only the neutral form of 1-hydroxypyrene partitions into the membrane and appreciable spectral changes are observed in the pH range of 9.0-11.5 in Tris-NaOH buffer. However, at pH 11 the spectral changes are maximum. The possibility of using 1-hydroxypyrene as a fluorescent molecular probe for lipid bilayer membranes in alkaline media has been examined, by employing fluorescence intensity and fluorescence anisotropy as probe parameters. The neutral form fluorescence intensity as well as fluorescence anisotropy is sensitive to the changes in the membrane properties and is capable of sensing the phase-transition. This is also capable of monitoring the changes in the membrane due to incorporation of cholesterol and the ethanol-induced interdigitation. The time resolved fluorescence data and the quenching experiments show that 1-hydroxypyrene occupies the water inaccessible interior of the liposome. The high anisotropy value of 1-hydroxypyrene in liposome suggests that it resides in a considerably rigid environment and is very sensitive to the temperature-induced changes in the liposome.
Fluorescence Behavior of the pH-Sensitive Probe Carboxy SNARF-1 in Suspension of Liposomes¶
When exposed to the intracellular environment fluorescent probes sensitive to pH exhibit changes of photophysical characteristics as a result of an interaction of the dye molecule with cell constituents such as proteins, lipids or nucleic acids. This effect is reflected in calibration curves different from those found with the same dye in pure buffer solutions. To study an interaction of the probe 5(and 6)-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7,1(3H)-isobenzofuran]-3one (carboxy SNARF-1) with membrane lipids, we measured its fluorescence in model systems of large unilamellar vesicles (LUV) prepared by extrusion. When the dye was removed from the bulk solution by gel filtration the relative fluorescence intensity of the lipid-bound dye form was enhanced, showing a strong interaction of the dye molecule with LUV membrane lipids. Surprisingly, the dye molecules seem to be bound predominantly to the outer surface of the lipid bilayer. The same situation was found with small unilamellar vesicles prepared by sonication. This effect makes it difficult to use carboxy SNARF-1 for measurements of the internal pH in suspensions of liposomes.