Real-time fluorescence polarization measurements: interaction of phospholipase A2 with a fluorescent lecithin derivative (original) (raw)
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Fluorescence polarization holds considerable promise for bioanalytical systems because it allows the detection of selective interactions in real time and a choice of fluorophores, the detection of which the biosample matrix does not influence; thus, their choice simplifies and accelerates the preparation of samples. For decades, these possibilities were successfully applied in fluorescence polarization immunoassays based on differences in the polarization of fluorophore emissions excited by plane-polarized light, whether in a free state or as part of an immune complex. However, the results of recent studies demonstrate the efficacy of fluorescence polarization as a detected signal in many bioanalytical methods. This review summarizes and comparatively characterizes these developments. It considers the integration of fluorescence polarization with the use of alternative receptor molecules and various fluorophores; different schemes for the formation of detectable complexes and the am...
A New Fluorescent Squaraine Probe for the Measurement of Membrane Polarity
Journal of Fluorescence, 2006
The present study was undertaken to evaluate the sensitivity of newly synthesized squaraine dye 1 to the changes in lipid bilayer physical properties and compared it with the well-known dye 2. Partitioning of the dye 1 into lipid bilayer was found to be followed by significant increase of its fluorescence intensity and red-shift of emission maximum, while intensity of the dye 2 fluorescence increased only slightly on going from aqueous to lipidic environment. This suggests that dye 1 is more sensitive to the changes in membrane properties as compared to dye 2. Partition coefficients of the dye 1 have been determined for the model membranes composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with positively charged detergent cetyltrimethylammonium bromide (CTAB), anionic phospholipid cardiolipin (CL), and sterol (Chol). The spectral responses of the dye 1 in different liposome media proved to correlate with the increase of bilayer polarity induced by Chol and CL or its decrease caused by CTAB. It was concluded that dye 1 can be used as fluorescent probe for examining membrane-related processes.
Journal of Biomedical Optics, 1997
The utility of 2Ј,7Ј-bis-(carboxyethyl)-5-(6Ј)-carboxyfluorescein (BCECF) for the execution of the structuredness of the cytoplasmic matrix (SCM) measurement for lymphocyte activation is investigated. Cells were incubated with BCECF/AM [2Ј,7Ј-bis-(carboxyethyl)-5(6Ј)-carboxyfluorescein acetoxymethylester], a nonfluorescent lipophilic acetoxymethylester that readily enters cells and is enzymatically hydrolyzed to fluorescent BCECF once inside. Leakage of BCECF out of cells is negligible in comparison to that observed with fluorescein, greatly reducing one source of background fluorescence. However, spontaneous hydrolysis of BCECF/AM in aqueous solution does contribute significant background fluorescence, which can be minimized by staining at relatively high concentrations of cells and subsequent dilution. As is the case with fluorescein, the polarization spectrum of intracellular BCECF shows a wavelength dependence not seen in the spectrum of the dye in homogeneous media of various viscosities. The more pronounced wavelength dependence of the polarization observed with BCECF compared with fluorescein suggests that BCECF might be preferable to fluorescein as a marker for the SCM test.
Fluorescence polarization of green fluorescence protein
Proceedings of The National Academy of Sciences, 2002
We report here the striking anisotropy of fluorescence exhibited by crystals of native green fluorescence protein (GFP). The crystals were generated by water dialysis of highly purified GFP obtained from the jellyfish Aequorea. We find that the fluorescence becomes six times brighter when the excitation, or emission, beam is polarized parallel (compared with perpendicular) to the crystal long axis. Thus, the major dipoles of the fluorophores must be oriented very nearly parallel to the crystal long axis. Observed in a polarizing microscope between parallel polars instead of either a polarizer or analyzer alone, the fluorescence polarization ratio rises to an unexpectedly high value of about 30:1, nearly the product of the fluorescence excitation and emission ratios, suggesting a sensitive method for measuring fluorophore orientations, even of a single fluorophore molecule. We have derived equations that accurately describe the relative fluorescence intensities of crystals oriented in various directions, with the polarizer and analyzer arranged in different configurations. The equations yield relative absorption and fluorescence coefficients for the four transition dipoles involved. Finally, we propose a model in which the elongated crystal is made of GFP molecules that are tilted 60° to align the fluorophores parallel to the crystal long axis. The unit layer in the model may well correspond to the arrangement of functional GFP molecules, to which resonant energy is efficiently transmitted from Ca2+-activated aequorin, in the jellyfish photophores.
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
Analysis of protein-ligand interactions by fluorescence polarization
Nature Protocols, 2011
Quantification of the associations between biomolecules is required both to predict and understand the interactions that underpin all biological activity. Fluorescence polarization (FP) provides a non-disruptive means of measuring the association of a fluorescent ligand with a larger molecule. We describe an FP assay in which binding of fluorescein-labelled inositol 1,4,5-trisphosphate (IP 3) to N-terminal fragments of IP 3 receptors can be characterised at different temperatures and in competition with other ligands. The assay allows the standard Gibbs free energy (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) changes of ligand binding to be determined. The method is applicable to any purified ligand-binding site for which an appropriate fluorescent ligand is available. FP can be used to measure low-affinity interactions in real-time without use of radioactive materials, it is non-destructive, and with appropriate care it can resolve ΔH° and ΔS°. The first part of the protocol, protein preparation, may take several weeks, while the FP measurements, once they have been optimised, would normally take 1-6 h.
Fluorescent probes used to monitor membrane interfacial polarity
Chemistry and Physics of Lipids, 1999
The polarity of the interface between a lipid bilayer membrane and bulk water is an important physical parameter of the membrane. It is likely that several membrane-dependent biological functions are modulated by this property. However, interfacial polarity can be difficult to define because of an imprecise knowledge of the molecular nature of the interface. Nevertheless, attempts have been made to measure this quantity with the use of fluorescent probes which are sensitive to the solvent polarity. Often, however, other factors, such as the rate of solvent relaxation must be known in order to interpret the fluorescent properties in terms of the dielectric constant. In addition, the spatial orientation and location of the fluorophore are often not known precisely. Nevertheless, there have been successful efforts to gain a more accurate knowledge of this aspect of membrane physical properties and its relationship to biological phenomena is discussed.
bioRxiv (Cold Spring Harbor Laboratory), 2023
Spatial discretization of biomolecules in the complex cellular environment is crucial for biomolecular form and function. The ability to better understand the driving force of spatial discretization of biomolecules in the complex cellular matrix remains a challenging task. We report on the robust polarity sensitive solvatochromic probe, FLAM, in conjunction with spectral phasor analysis as a general method for studying environmental polarity in biological systems. We find that phase separated proteins of SFPQ have distinct polarity depending on the type of phase separation occurring, suggesting that polarity plays a role in the formation of phase separated condensates. When using FLAM in cells, distinct subcellular environmental polarity distribution but similar trend of changes is observed for cells under similar type of stressors. Taken together, our method puts forth an exciting development in the tool set for the study of phase separation.
Simultaneous Detection of Local Polarizability and Viscosity by a Single Fluorescent Probe in Cells
Biophysical journal, 2018
Many intracellular reactions are dependent on the dielectric ("polarity") and viscosity properties of their milieu. Fluorescence imaging offers a convenient strategy to report on such environmental properties. Yet, concomitant and independent monitoring of polarity and viscosity in cells at submicron scale is currently hampered by the lack of fluorescence probes characterized by unmixed responses to both parameters. Here, the peculiar photophysics of a green fluorescent protein chromophore analog is exploited for quantifying and imaging polarity and viscosity independently in living cells. We show that the polarity and viscosity profile around a novel hybrid drug-delivery peptide changes dramatically upon cell internalization via endosomes, shedding light on the spatiotemporal features of the release mechanism. Accordingly, our fluorescent probe opens the way to monitor the environmental effects on several processes relevant to cell biochemistry and nanomedicine.