TRANES Spectra of Fluorescence Probes in Lipid Bilayer Membranes: An Assessment of Population Heterogeneity and Dynamics

Time-resolved fluorescence of eight fluorescence probes were studied in EggPC bilayer membrane vesicles. Emission wavelength dependent fluorescence decays were analyzed in a model-independent way to obtain time resolved area normalized emission spectra (TRANES). The TRANES spectra of the probes studied were classified into four types: (1) spectra that are identical at all time (one emissive species), (2) spectra that show an isoemissive point (two emissive species), (3) spectra that shift continuously with time (slow solvation dynamics or multiple species), and (4) spectra that shift for a short time and thereafter one or two emissive species are indicated. The TRANES spectra of these eight probes, except RH421, belong to the type 1, 2, or 4. The continuous shift of the TRANES spectra that was observed for the probe RH421 is attributed to multiple ground state species and not due to slow solvation dynamics.     

Study of plasma membrane heterogeneity using a phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene [2-(3-(diphenylhexatriene)propanoyl)-3-palmitoyl-l-α-phosphatidylcholine]

The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH derivatives have been used to characterize structural and physicochemical properties of specific membrane domains. Steady-state and fluorescence decay measurements of three probes, DPH (1,6-diphenyl-1,3,5-hexatriene), TMA-DPH [1-(4-trimethyl-ammonium-phenyl)-6-phenyl-1,3,5-hexatriene], and a phosphatidylcholine derivative of DPH, DPH-pPC [2-(3-(diphenylhexatriene)propanoyl)-3-pamitoyl-L--phosphatidyl choline], have been performed in erythrocyte membranes and in lymphocyte plasma membranes. The steady-state fluorescence polarization of the three probes showed a similar trend in both membranes. In fact either in erythrocyte or in lymphocyte plasma membranes the fluorescence polarization values of DPH-pPC and TMA-DPH were similar, but significantly higher with respect to DPH. A better characterization of erythrocyte and lymphocyte plasma membranes was possible by using fluorescence decay measurements. The data suggest the possible use of different DPH derivatives to characterize specific domains in biological membranes.     

An interaction of helicid with liposome biomembrane

An interaction of helicid with phosphatidylcholine liposome biomembrane was studied by transmission electron microscopy, UV-vis, fluorescence, Raman and ³¹P NMR spectra. The results indicate that most of helicid molecules associate with liposomes at their surface and some of them penetrate the liposomes and locate in the hydrophobic regions of the membrane. The distribution coefficient K_D between liposome phases and aqueous phases is 13.5. The liposome becomes more dispersive and stable in the presence of helicid. The microenvironmental micropolarity and the microhydrophobicity of liposome membrane decrease with the increase of helicid concentration. The interaction of helicid molecules with liposome results in a slight decrease of the membrane longitudinal order, and an increase of the membrane lateral order. A model for the interaction of helicid with liposome biomembrane is proposed on the basis of the change of microenvironment parameters of liposome including the micropolarity, microhydrophobicity and membrane order. The change of microenvironment parameters results mainly from hydrogen bonding interaction between the hydroxyl groups of the pyranoside rings of helicid molecules and the polar head groups of phosphatidylcholine.     

Structural Changes in Lymphocytes Membrane of Chernobyl Clean-up Workers from Latvia

ABM (3-aminobenzanthrrone derivative) developed at the Riga Technical University, Riga, Latvia) has been previously shown as a potential probe for determination of the immune state of patients with different pathologies .The fist study (using probe ABM) of peripheral blood mononuclear cells (PBMC) membranes of 97 Chernobyl clean-up workers from Latvia was conducted in 1997. Now we repeatedly examine the same (n = 54) individuals in dynamics. ABM spectral parameters in PBMC suspension, fluorescence anisotropy and blood plasma albumin characteristics were recorded. In 1997 screening showed 5 different patterns of fluorescence spectra, from which in 2007 we obtained only two. These patterns of spectra had never been previously seen in healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc., examined by us. Patterns of ABM fluorescence spectra are associated with membrane anisotropy and conformational changes of blood plasma albumin. We observed that in dynamics 1997–2007 the lipid compartment of the membrane became more fluid while the lipid-protein interface became more rigid. The use of probe ANS and albumin auto-fluorescence allowed show conformational alterations in Chernobyl clean-up workers blood plasma. It is necessary to note that all investigated parameters significantly differ in observed groups of patients. These findings reinforce our understanding that that the cell membrane is a significant biological target of radiation. The role of the membrane in the expression and course of cell damage after radiation exposure must be considered. So ten years dynamic of PBMC membrane characteristics by ABM (spectral shift and anisotropy indexes) in Chernobyl clean-up workers reveal progressive trend toward certain resemblance with those of chronic B-cell lymphoid leukemia.     

Dipole Moments of Phenylnaphthylamine Fluorescence Probes and Study of Dielectric Interactions in Human Erythrocyte Ghosts

Electrooptical absorption and emission methods were used to measure the dipole moments of 1-phenylnaphthylamine (1-AN) and 1,8-anilinonaphthalene sulfonate (1,8-ANS) fluorescence probes in the ground and excited states. The measurements were performed in cyclohexane (1-AN) and dioxane (1-AN and 1,8-ANS). Our results show that the charge distribution in 1-AN differs substantially from that in 1,8-ANS. The spectral dependence of electrooptical coefficients in the case of 1,8-ANS indicates that the absorption band is a superposition of several (at least two) electronic transitions. We found that the electronic spectra of 1-AN in erythrocyte ghosts are inhomogeneously broadened. Due to inhomogeneous broadening it is possible to excite selectively molecules located at different sites. Dielectric interactions (given by the dielectric constant) were investigated from the position of the maximum of the fluorescence quantum spectra of 1-AN. The dielectric constant of human erythrocyte ghosts varies from about 6.7 ± 0.8 to about 17.6 ± 3.5, depending on the excitation frequency and, consequently, on the location of the probe.     

Solvent dependent study of carbonyl vibrations of 3‐phenoxybenzaldehyde and 4‐ethoxybenzaldehyde by Raman spectroscopy and ab initio calculations

A Raman spectroscopy investigation of the carbonyl stretching vibrations of 3‐phenoxybenzaldehye (3Phbz) and 4‐ethoxybenzaldeheyde (4Etob) was carried out in binary mixtures with different polar and nonpolar solvents. The purpose of this study was twofold: firstly, to describe the interaction of the carbonyl groups of two solute molecules in terms of a splitting in the isotropic and anisotropic components and secondly, to analyze their spectroscopic signatures in a binary mixture. Changes in wavenumber position, variation in the anisotropic shift and full width half maximum were investigated for binary mixtures with different mole fractions of the reference systems. In binary mixtures, the observed increase in wavenumber with solvent concentration does not show linearity, indicating the significant role of molecular interactions on the occurrence of breaking of the self‐association of the solute. In all the solvents, a gradual decrease in the anisotropic shift reflects the progressive separation of the coupled oscillators with dilution. Γ_i(η_c), 3Phbz—solvent mixtures, exhibit a gradual decrease with decrease in the concentration of the solute which is an evidence on the influence of micro viscosity on linewidth. For 4Etob, the carbonyl stretching vibration shows two well‐resolved components in the Raman spectra, attributed to the presence of two distinct carbonyl groups: hydrogen‐bonded and free carbonyl groups. The intensity ratio of the carbonyl stretching vibration of these two types of carbonyl groups is studied to understand the dynamics of solute/solvent molecules owing to hydrogen bond interactions. Ab initio calculations were employed for predicting relevant molecular structures in the binary mixtures arising from intermolecular interactions, and are related to the experimental results. Copyright © 2009 John Wiley & Sons, Ltd.     

Rotational Diffusion of Coumarins in Alcohols: A Dielectric Friction Study

The rotational dynamics of three structurally similar polar molecules viz., coumarin 440, coumarin 151 and coumarin 450 has been studied in alcohols at room temperature using steady-state fluorescence depolarization method and time correlated single photon counting technique. The observed reorientation times of all the three probes are found to be longer than those predicted by Stokes–Einstein–Debye (SED) hydrodynamic theory with stick boundary condition and a deviation towards super-stick behavior is noted. Dielectric friction theories of Nee–Zwanzig and van der Zwan–Hynes, which treat the solute as a point dipole, overestimate the dielectric friction contribution exhibited by all the three coumarins in alcohols. Results are discussed in the light of theoretical models and the possibility of hydrogen bonding between the amino group of the probe molecules and the hydroxyl group of the alcohols.     

Modulation of Fluorophore Environment in Host Membranes of Varying Charge

The net electrical charge of the biological membrane represents an important parameter in the organization, dynamics and function of the membrane. In this paper, we have characterized the change in the microenvironment experienced by a membrane-bound fluorescent probe when the charge of the phospholipids constituting the host membrane is changed from zwitterionic to cationic with minimal change in the chemical structure of the host lipid. In particular, we have explored the difference in the microenvironment experienced by the fluorescent probe 2-(9-anthroyloxy)stearic acid (2-AS) in model membranes of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cationic 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC) which are otherwise chemically similar, using the wavelength-selective fluorescence approach and other fluorescence parameters. Our results show that the microenvironment experienced by a membrane probe such as 2-AS is different in POPC and EPOPC membranes, as reported by red edge excitation shift (REES) and other fluorescence parameters. The difference in environment encountered by the probe in the two cases could possibly be due to variation in hydration in the two membranes owing to different charges.     

Use of the red-edge excitation effect for investigation of dielectric interactions in biomembranes

Dipole moments of the fluorescent probes 1-phenylnaphthylamine (1-AN) and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) are measured using electro-optical absorption and emission methods. Dipole moments in the ground and excited states were measured in cyclohexane and dioxane. It is shown that the charge distributions in the 1-AN and 1,8-ANS molecules differ substantially. The spectral dependence of the electro-optical coefficients suggests that the absorption spectrum of 1,8-ANS is due to a superposition of (at least two) electronic transitions. It is found that spectra of 1-AN in erythocyte ghosts are inhomogeneously broadened. The above effect makes it possible to selectively excite probe molecules localized at different sites of a membrane. Dielectric interactions (described by the local dielectric constant) are investigated in human erythrocyte membranes. It is found that the dielectric constant of erythrocyte membranes varies from 6.79 ±0.8 to 17.6±3.5 depending on the excitation frequency and, therefore, on the localization of the probe. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 66, No. 3, pp. 387–393, May–June, 1999.     

Laurdan in Fluid Bilayers: Position and Structural Sensitivity

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41°C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel–fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.     

Study on fluorescence spectra of molecular association of acetic acid-water

Fluorescence spectra of acetic acid-water solution excited by ultraviolet （UV） light are studied, and the relationship between fluorescence spectra and molecular association of acetic acid is discussed. The results indicate that when the exciting light wavelength is longer than 246 nm, there are two fluorescence peaks located at 305 and 334 nm, respectively. By measuring the excitation spectra, the optimal wavelengths of the two fluorescence peaks are obtained, which are 258 and 284 nm, respectively. Fluorescence spectra of acetic acid-water solution change with concentrations, which is primarily attributed to changes of molecular association of acetic acid in aqueous solution. Through theoretical analysis, three variations of molecular association have been obtained in acetic acid-water solution, which are the hydrated monomers, the linear dimers, and the water separated dimers. This research can provide references to studies of molecular association of acetic acid-water, especiMly studies of hydrogen bonds.     

407 nm 光诱导的红细胞荧光光谱随浓度增加红移的机理分析

用407nm光激发不同浓度离体红细胞可以产生较强的荧光光谱,进一步研究发现这些荧光光谱中存在两个明显的、频谱宽度大致相等的荧光发射区,而且在生理盐水中随着红细胞浓度的增加,其发射的荧光光谱的谱峰值位置向长波方向移动,即出现“红移”现象.从理论上对这种“红移”现象的产生机理进行了分析,表明这种现象是由于浓度变化对荧光团电子能级产生的微扰发生变化所致.该研究结果对现代医学中的荧光光谱诊断技术和低功率激光照射疗法都有一定的参考价值.     

4-Aminophthalimide Derivatives as Environment-Sensitive Probes

The potential of 4-aminophthalimide (AP) and its derivatives as fluorescence probes for organized media is highlighted. The fluorescence response of AP, as measured from the position of the fluorescence maximum, fluorescence intensity and lifetime, is highly sensitive to the polarity of the medium. The sensitivity of the fluorescence parameters is further enhanced due to the involvement of the emitting intramolecular charge transfer state in hydrogen bonding interaction with the solvent molecules containing hydroxyl groups. It is shown that the microheterogeneous environments of organized media such as cyclodextrins and micelles can be very conveniently monitored using this probe. The results of the investigations carried out employing AP and its derivatives as fluorescence probe molecules in these media clearly suggest that a combination of the hydrophobic interaction with the host media and hydrogen bonding interaction with the solvent molecules determine the location of the fluorophore, which in all cases is found to be the interfacial region separating the nonpolar core of the micelle or the cyclodextrin cavity and the polar aqueous environment. Guidelines for the design of probes of this class of systems for the nonpolar core region of the micelles are provided and possible ways to increase the sensitivity of the fluorescence response of the systems are suggested.     

MONITORING OF CURING PROCESS BY FLUORESCENCE TECHNIQUE. FLUORESCENCE PROBE AND LABEL BASED ON 5-DIMETHYLAMINONAPHTHALENE-1-SULFONAMIDE DERIVATIVES (DNS)

The curing reaction of glycidyl ether bisphenol A (DGEBA) with n-butyl amine and/or N-methylethylenediamine was monitored by fluorescence spectroscopy. 5-Dimethylaminonaphthalene-1-sulfonamide (DNS) fluorophore was used as a probe and/or label. Fourier transform infrared (FTIR) analysis revealed that the rate constant for the addition reaction of the primary amino group hydrogen of n-butylamine to the epoxide ring is more than four times larger than that arising from a secondary amine. Significant differences have been observed between the fluorescence behavior of the DNS as a probe and label, especially in the system DGEBA–n-butyl amine. Integrated fluorescence intensity for the DNS label, in contrast to the DNS probe, indicates the most important changes in chemical transformations of this reaction mixture (the onset of tertiary amino groups and maximum concentration of secondary amino groups). Similarly, the dependence of the half-bandwidth on the epoxy groups conversion for the DNS label shows these stages of the curing reaction as well. In the system DGEBA–N-methylethylenediamine, the reactivity of the secondary amino group hydrogen is higher than that of the primary amino group. A change in slope of the dependence of integrated fluorescence intensity on epoxy group conversion clearly indicates the gel point and entry of the system into the glassy state. The DNS probe does not sense any of these changes. From the emission spectra of the DNS probe and/or label, the average value <v> = ΣI_F (ν)ν/ΣI_F (ν) of the emission band position has been correlated with the epoxy group conversion determined by FTIR. Smooth dependencies were obtained in all cases. This enables one to monitor on line and in real time the epoxy group conversion.     

Structure and Luminescent Properties of the 4-Arylidene-2-Aryl-5-Oxazolones(Azlactones)In Solution and Crystalline State

Experimental and theoretical evaluations have proven that very low fluorescence quantum yields of azlactones in solution are not caused by an efficient inter system crossing from S₁*(ππ) to T*(nπ) states, but rather from solvation and steric effects, that result in non-planarity of the molecular system. High fluorescence quantum yields in the solid state are attributed to the planarity of the azlactone molecule upon packing into the crystal lattice. Supporting evidence was found upon observation of the excited state proton transfer (ESIPT) bands of fluorescence emissions of o-hydroxyarylidene derivatives. The photoinstability of azlactones in liquid states are attributed to photochemical E-Z isomerization and cleavage of the hetero ring α to the carbonyl group.     

Fluorescence Investigation on the Interaction of a Prevalent Competitive Fluorescent Probe with Entomic Odorant Binding Protein

In recent years, one prevalent competitive fluorescent probe, N-phenyl-1-naphthylamine (1-NPN), was frequently utilized to measure the binding affinity of entomic odorant binding proteins (OBPs) with diverse plant volatiles or pheromones. Nevertheless, the details and model of the binding interaction are still largely unknown, although it is vital to investigate the physiological function of OBPs. Here we studied the binding interaction between 1-NPN and OBP2, a recombinant OBP from eastern honeybee, Apis cerana, by the combination of fluorescence quenching spectra, synchronous fluorescence spectra, ultraviolet spectra, circular dichroism spectra, and molecular docking. The Stern–Volmer curve of the fluorescence quenching of OBP2 by 1-NPN indicated it was a static quenching mechanism, and the binding constants and binding number were determined, respectively. Based on the Förster theory of nonradiation energy transfer (FRET), the binding distance was calculated, and the intrinsic fluorescent energy was predicted to transfer from the donor OBP2 to the acceptor 1-NPN. Synchronous fluorescence spectra and circular dichroism spectra were used to investigate the conformational change in binding progress. The thermodynamic parameters showed that the interaction was mainly driven by hydrophobic force, which was validated by the molecular docking; meanwhile, the binding mode was revealed and one hydrogen bond was found between the nitrogen atom of 1-NPN and Glu29 of OBP2.     

激光诱导荧光光谱法研究血细胞衰变规律

用激光诱导荧光光谱法研究了全血溶液在不同衰变时间的荧光光谱变化规律.经小白鼠眼眶取血后配成不同浓度的全血溶液,每隔三小时检测一次其荧光光谱,得到了全血溶液在整个衰变过程中不同时间段的荧光光谱.研究结果表明：存放在室温空气中的血液会随存放时间的延长,其628nm处的荧光峰产生红移,同时荧光峰强度也随之减弱.提出血液荧光光谱峰值红移是由于血细胞在老化过程中红细胞膜不同程度受损引起的.红细胞受损导致溶血,其中的血红蛋白大分子之间将发生共振能量转移,引起自吸收,从而使荧光光谱强度降低.其研究结果将会对研究血液细胞的衰变机理,理解机体细胞的衰变具有一定的参考意义.     

Membrane Dipole Potential as Measured by Ratiometric 3-Hydroxyflavone Fluorescence Probes: Accounting for Hydration Effects

We previously applied the electrochromic modulation of excited-state intramolecular proton-transfer (ESIPT) reaction for the design of novel 3-hydroxyflavone (3-HF) derivatives as fluorescent probes for measuring the dipole potential, Ψ_D, in lipid bilayers (Klymchenko et al., Proc. Natl. Acad. Sci. USA, 2003, 100, 11219). In the present work, this method was revisited to take into account the influence of the bilayer hydration on the emission ratiometric response of 3-HF probes. For this reason, it was necessary to deconvolute the whole fluorescence spectra into three bands corresponding to the non H-bonded forms, normal N^* and tautomer T^* forms, both participating to the ESIPT reaction, and to the H-bonded H–N^* form, excluded from this reaction. This allowed us to determine the pure N^*/T^* intensity ratio, without any contribution from the H–N^* form emission depending essentially on the bilayer hydration. This new approach allowed us to confirm the correlation we obtained between the response of 3-HF probes on dipole potential modifications and the corresponding response of the reference fluorescent probe di-8-ANEPPS, thus further confirming the potency of 3-HF probes as excellent emission ratiometric probes to measure dipole potential in lipid membranes.     

Fluorescence Studies of the Acetylcholine Receptor: Structure and Dynamics in Membranes and Cells

The nicotinic acetylcholine receptor (AChR) is the archetype member of the superfamily of ligand-gated ion channels that mediate fast intercellular communication in response to endogenous neurotransmitters. Here I review a series of biophysical studies on the AChR protein, with particular focus on the interactions of the macromolecule with its lipid microenvironment. Fluorescence recovery after photobleaching and phosphorescence anisotropy studies of the membrane-embedded AChR have contributed to our understanding of the translational and rotational dynamics of this protein in synthetic lipid bilayers and in the native membrane. Electron spin resonance studies led to the discovery of a lipid fraction in direct contact with the AChR with rotational dynamics 50-fold slower than that of the bulk lipids. This lipid belt region around the AChR molecule has since been intensively studied with the aim to define its possible role in the modulation of receptor function. The polarity and molecular dynamics of solvent dipoles—mainly water—in the vicinity of the lipids in the AChR membrane have been studied exploiting the amphiphilic fluorescent probe Laurdan's exquisite sensitivity to the phase state of the membrane, and Förster-type resonance energy transfer (FRET) was introduced to characterize the receptor-associated lipid microenvironment. FRET was used to discriminate between the bulk lipid and the lipid belt region in the vicinity of the protein. Further refinement of this topographical information was provided by the parallax method using phospholipid spin labels. The AChR-vicinal lipid is in a liquid-ordered phase and exhibits a higher degree of order than the bulk bilayer lipid. Changes in FRET efficiency induced by fatty acids, phospholipid, and cholesterol also led to the identification of discrete sites for these lipids on the AChR protein. I also illustrate the extension of Laurdan fluorescence studies to intact living cells heterologously expressing AChR in a brief section devoted to recent studies using two-photon fluorescence microscopy. The spatial resolution afforded by the two-photon optical sectioning of the cell in combination with the advantageous spectroscopic properties of Laurdan are exploited to obtain information on the physical state of the lipid environment of the membrane. Finally, the application of site-specific labeling and steady-state fluorescence spectroscopy to probe the location of AChR membrane-embedded domains is illustrated. The topography of the pyrene-labeled Cys residues in transmembrane domains M1, M4, M1, and M4 with respect to the membrane was determined by differential fluorescence quenching with lipid-resident spin-labeled probes. Cys residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear -helical structure, but not so for M1, for which classical models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of nonhelical structure and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.