Spectral analyses of extrinsic fluorescence of the nerve membrane labeled with aminonaphthalene derivatives

A fluorescence spectrophotometer was constructed to determine the emission spectrum of a nerve labeled with various fluorochromes. Using this spectrophotometer, the spectra of 2-p-toluidinylnaphthalene 6-sulfonate (2,6-TNS) and other aminonaphthalene derivatives in squid giant axons were determined at the peak of nerve excitation, as well as in the resting state of the axons. During nerve excitation the fluorescent light deriving from the 2,6-TNS-stained nerve undergoes a transient change in intensity. The spectrum of the light contributing to this change in intensity was found to be much narrower and sharper than the fluorescent spectrum of the light arising from labeled axons at rest. This narrow and sharp spectrum is interpreted as being derived from a transient variation in the polarity of the 2,6-TNS binding sites in the axon. In the Appendix, the results of a physicochemical investigation into the factors affecting the fluorescence of 2,6-TNS in vitro are described.     

In vitro capacitation of bull spermatozoa by oviductal fluid and its components

Sperm capacitation is crucial for fertilization. However, debate continues on exactly how, where and when capacitation is elicited in the bovine female genital tract. In this study we used merocyanine-540 and the chlortetracycline (CTC) assay to test how capacitation of bull spermatozoa is affected in vitro by exposure to oviductal fluid (ODF) collected in vivo, various glycosaminoglycans (GAGs) or bicarbonate. Following different durations of exposure, spermatozoa were stained with CTC or merocyanine-540, and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Incubation time did not significantly affect capacitation. Exposure (30-120 min) to ODF capacitated (p < 0.05) bull spermatozoa as measured by either merocyanine-540 or CTC. Hyaluronan was the only GAG that induced a significant increase in B-pattern spermatozoa (capacitated; p = 0.012) compared with controls. Dermatan sulphate also induced capacitation (merocyanine-540 high fluorescence; p = 0.035). Exposure to bicarbonate-enriched media also yielded an increase in merocyanine-540 high fluorescence (p < 0.0001). When bicarbonate was added to the other treatments (ODF or GAGs) an equal increase in merocyanine-540 high fluorescence was noted (p < 0.0001), compared with before addition of bicarbonate and independent of the treatment before exposure. There was no significant difference in the number of B-pattern spermatozoa when bicarbonate was added, but an significant increase in spermatozoa with an acrosome-reacted (AR)-pattern (p < 0.0001) was observed. Exposure of spermatozoa to solubilized zonae pellucidae significantly increased the AR-pattern spermatozoa (p = 0.016). In conclusion, ODF was more potent in inducing capacitation of bull spermatozoa than the individual GAGs. Our results also indicate that bicarbonate is an effector of bull sperm capacitation.     

Fluorescence of squid axon membrane labelled with hydrophobic probes

Summary Extrinsic fluorescence changes in squid giant axons were examined under a variety of experimental conditions using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and other fluorescent probes. Measurements of the degree of polarization of the fluorescent light (with the axis of the polarizer parallel to the longitudinal axis of the axon) indicated that the class of the TNS molecules in the axon membrane which participate in production of fluorescence signals have a definite orientation with their absorption and emission oscillators directed parallel to the long axis of the axon. Rectangular depolarizing voltage pulses produced a transient decrease in the fluorescent intensity, of which the early component is correlated tentatively with the rise in the membrane conductance. In response to hyperpolarizing pulses, there was an increase in fluorescence intensity which may be explained in terms of increased incorporation of TNS into the ordered structure in the membrane. Hyperpolarizing responses in KCl depolarized axons were accompanied by a change in fluorescent intensity. Tetrodotoxin appeared to suppress the initial component of the fluorescence signal produced by depolarizing clamping pulses. The technique for detecting these fluorescence changes and the physico-chemical properties of TNS are described in some detail.     

The components of merocyanine-540 absorption spectra in aqueous, micellar and bilayer environments.

Spectral-data-processing and curve-fitting techniques have been applied to the decomposition of merocyanine-540 absorption spectra in aqueous, micellar and bilayer environments. The various resolved component bands have been assigned to dye monomers, dimers, or larger aggregates, either in polar or non-polar environments. The analysis of spectral parameters (lambda max and integrated intensity) of the overall spectra and of each component has revealed that merocyanine 540 is a useful probe in studies of membrane structure and dynamics using visible-absorption spectroscopy. In particular, the monomer lambda max and the integrated intensity, i.e. area, of the dimer population are very useful in this respect. The monomer lambda max is especially sensitive to polarity changes and is thus useful, e.g. in the precise determination of critical micellar concentrations. The fractional area of the dimer increases with the packing density of the phospholipid-hydrocarbon region near the interface and is thus very sensitive to changes in vesicle curvature and to the presence of sterols or intrinsic polypeptides in the bilayer.     

Fluorescent styryl dyes applied as fast optical probes of cardiac action potential

Several styryl dyes were tested as fast optical probes of membrane action potentials in mammalian heart muscle tissue. After staining, atrial specimens were superfused in physiological salt solution, and fluorescence was excited by an argon ion laser. Excitation spot size on the surface of the preparation was 60 m in diameter. Dyes RH 160, RH 237, and RH 421 performed excellently as fast fluorescent probes of cardiac membrane potential. Fractional fluorescence changes, F/F, due to the action potential were in the range 2 to 6% at 514.5 nm excitation. Rise times of the action potential onset detected with each of the dyes were less than 0.5 ms, which is as fast or even faster than microelectrode measurements (atria of the rat). Thus membrane potential changes could be monitored with high resolution in both time and space. Emission spectra from heart muscle preparations stained with these dyes were shifted to shorter wavelengths by 70 nm and more as compared to spectra of the dyes in ethanol solution. The fluorescence spectrum of RH 160 at resting potential and the spectrum recorded during the plateau phases of the action potential were measured and showed no difference within the spectral resolution. As can be concluded from measurements of fluorescence changes at different excitation wavelengths, electrochromism cannot be the only mechanism causing the potential response.     

Effect of PAF on polyrnorphonuclear leucocyte plasma membrane polarity: a fluorescence study

The effect of PAF on the plasma membrane polarity of polymorphonuclear leukocytes (PMNs) was investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-1auroyl) naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surrounding. Laurdan shows a marked steady-state emission blue-shift in non-polar solvents, with respect to polar solvents. Our results demonstrate that PAF (10(-7) M) induces a blue shift of the fluorescence emission spectra of Laurdan. These changes are blocked in the presence of the PAF antagonist, L-659,989. Our data indicate that the interaction between PAF and PMNs is accompanied by a decrease in polarity in the hydrophobic-hydrophilic interface of the plasma membrane.     

Recording changes in extracellular pH via confocal microscopy during generation of excitation potentials in higher plants

The method of confocal microscopy was applied to pH changes recorded in the apoplast of the stem of a pumpkin seedling during the generation of the action potential and variation potential. To record the change in pH, the fluorescent probe FITC-dextran was used. An analysis of the obtained fluorescent images and the determination of the fluorescence spectra showed that FITC-dextran is located in the stemcell walls. The propagation of the action and variation potentials is accompanied by a transient increase in the probe fluorescence intensity, which indicates the alkalization of cell walls. This transient alkalization is suggested to be due to the temporary inactivation of electrogenic H⁺-pump in the plasma membrane.     

Monitoring Biophysical Properties of Lipid Membranes by Environment-Sensitive Fluorescent Probes

We review the main trends in the development of fluorescence probes to obtain information about the structure, dynamics, and interactions in biomembranes. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Progress is being made in increasing the information content and spatial resolution of the probe responses. Multichannel environment-sensitive probes that can distinguish between different membrane physicochemical properties through multiple spectroscopic parameters show considerable promise.     

Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation

We investigated the effect of supplementing extended boar semen with different amounts of hyaluronan (HA) prior to freezing on post-thaw sperm characteristics. Using a split sample design, the effect of HA at a final concentration of 500 or 1000 microg/ml semen on post-thaw motility parameters, and membrane lipid architecture status assessed by merocyanine-540/YOPRO-1 and flow cytometry were evaluated. HA-supplementation improved motility parameters (P < 0.05 to P < 0.001) and decreased the percentage of hyperactivated spermatozoa (P < 0.05). HA-supplemented samples had more spermatozoa showing high lipid membrane stability as assessed with merocyanine-540. In conclusion, HA appeared to preserve post-thaw spermatozoa viability in vitro and maintained membrane stability after cryopreservation.     

A fluorimetric study of Mg2]nduced structural changes in thylakoid membrane protein.

Low concentrations of Mg²⁺ (concn < 10 mm) generate structural changes in delipidated spinach chloroplast lamellae, that appear as changes in the fluorescence yield of native tryptophyl residues and of the externally added polarity probe magnesium 1-anilinonaphthalene-8-sulfonate.The delipidated lamellae, consisting essentially of structural protein monomers and aggregates, bind magnesium 1-anilinonaphthalene-8-sulfonate to the extent of 126 ± 13 nmol/mg protein, and with a dissociation constant K_D = 167 μM. Bound ANS fluoresces at 458 nm with a quantum yield Φ = 0.121. Tryptophyls sensitize the fluorescence of bound ANS with a maximal efficiency T_max = 0.85. Assuming completely random orientation of the interacting chromophores, an interchromophore separation $\text{R = 17.3}\text{A}\text{?}$ is calculated. Only two-thirds of the membrane tryptophyls have ANS-binding sites in their vicinity.Mg²⁺ binds to the delipidated membranes with a dissociation constant K_D = 2 mM. The binding is attended by enhancement of magnesium 1-anilinonaphthalene-8-sulfonate fluorescence, and deenhancement of tryptophyl fluorescence, while the efficiency of interchromophore excitation transfer increases only slightly. These effects suggest that Mg²⁺ generates a structural change which lowers the polarity of the membrane region where tryptophyl and magnesium 1-anilinonaphthalene-8-sulfonate are situated, but which has a minor effect only on the interchromophore separation.     

Heparin and dermatan sulphate induced capacitation of frozen-thawed bull spermatozoa measured by merocyanine-540

Glycosaminoglycans (GAGs) are present in the oviduct in which the major part of sperm capacitation occurs. In this study we have tested how capacitation of frozen-thawed bull spermatozoa is effected by exposure to different GAGs detectable or possibly present in oviductal fluid; i.e. heparin, hyaluronan, heparan sulphate, dermatan sulphate and chondroitin sulphate. Following exposure of different duration, the spermatozoa were stained with either Chlortetracycline (CTC) or merocyanine-540 and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Heparin elicited a significant increase in the number of alive, capacitated spermatozoa, either expressed as higher merocyanine-540 fluorescence (p < 0.0001) or as B-pattern (p = 0.0021) in the CTC assay, during 4 h of incubation. When comparing the different GAG treatments one by one to the negative control in the flow cytometric study, only heparin and dermatan sulphate were significant (p < 0.0001) higher than the control at 0-30 min of incubation. Duration of incubation did not affect the proportion of capacitated spermatozoa when measured as merocyanine-540 fluorescence or CTC B-pattern, but the length of the incubation did affect the number of dead (Yo-PRO 1 positive) spermatozoa (p < 0.0001). Exposure to zona pellucida proteins significantly increased the proportion of acrosome reacted spermatozoa (p = 0.016). Both heparin and dermatan sulphate induce capacitation of frozen-thawed bull spermatozoa in vitro.     

Kinetics of the association of potential-sensitive dyes with model and energy-transducing membranes: Implications for fast probe response times

Summary The second-order rate constants characterizing the association of potential-sensing dyes of the cyanine, merocyanine, and oxonol classes with glycerylmonooleate suspensions, azolectin vesicles, or submitochondrial particles have been measured and the implications for redistribution type mechanisms proposed to explain the potential-dependent optical signals of these probes considered. The second-order rate constants obtained for the cyanines and oxonols are compatible with microsecond probe response times only on the assumption that a high local dye concentration exists in the aqueous phase immediately adjacent to the membrane surface. Calculations based on a surface charge density induced by a bias potential suggest that the necessary local concentration cannot be attained by a diffusion polarization mechanism. A model based on the rapid recombination of ejected dye with the membrane bilayer seems capable of explaining microsecond probe response times in systems where the potential is rapidly changing polarity; calculations suggest that an ejected dye molecule would not diffuse out of an unstirred layer of 100 microns thickness on a millisecond time scale. Microsecond probe responses are also compatible with a first-order potential-dependent dye ejection from the membrane with no rapid recombination when the potential is not changing polarity. The apparent first-order rate constants describing the interaction of merocanine M-540 with a glycerylmonooleate suspension are independent of dye concentration; the reaction may be diffusion limited. The high local dye concentration need not be met in this case for a mechanism based on the transfer of dye onto the membrane from the aqueous phase to describe the microsecond signals of this dye, but other mechanisms have been proposed to explain such signals. The mechanism leading to potentialdependent signals from optical probes appear to differ substantially between suspensions of energy-transducing biological membranes and those involving excitable membranes such as the squid giant axon or model black lipid membranes.     

Fluorescent lipid probes in the study of viral membrane fusion

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion.     

Changes in the lipid and protein components of thymus cell membrane during lipid peroxidation]

Nonenzymatic lipid peroxidation in thymus cell plasma membranes was studied. The composition of lipid and protein components, intensity of fluorescence of the membrane probes (1-anilinonaphthalene-8-sulfonate, 4-dimethylaminochalcon, eosin, pyronin and rhodamine), fluorescence polarization of tryptophan residues of membrane proteins and quenching by acrylamide of intrinsic fluorescence of proteins were determined. Induction of lipid peroxidation by the Fe(2+)-ascorbate system caused changes in the composition and structure of lipids. This was paralleled with changes in the structural-dynamic organization of membrane proteins, transition of some peripheral proteins to the water phase and increased solubilization of integral proteins by Triton X-100.     

Erythrocyte membranes - compression of lipid phases by increased cholesterol content

Lipid and protein interactions were studied in guinea pig erythrocytes containing a normal or a two-fold increased amount of cholesterol. The electron spin resonance (ESR) spectra of cholesterol-loaded cells labeled with fatty-acid probes showed an increase in the local viscosity of the membrane as compared with control cells. This increase reflects changes in the interior of the lipid matrix of the membrane because the probes resisted destruction by ascorbate, were unaffected by the action of pronase, and gave spectra similar to those of liposomes. No differences were observed between control and cholesterol-loaded cells in the conformation of the membrane proteins by either the infrared spectra or the ESR spectra of cells labeled with maleimide probes.     

Voltage-dependent translocation of R18 and DiI across lipid bilayers leads to fluorescence changes.

We show that the lipophilic, cationic fluorescent dyes R18 and Dil translocate from one monolayer of a phospholipid bilayer membrane to the other in a concentration and voltage-dependent manner. When the probes were incorporated into voltage-clamped planar membranes and potentials were applied, displacement currents resulted. The charged probes sensed a large fraction of the applied field. When these probes were added to only one monolayer, displacement currents were symmetrical around 0 mV, indicating that the probes distributed equally between the two monolayers. Charge translocation required that the bilayer be fluid. When membranes were in a condensed gel phase, displacement currents were not observed; raising the temperature to above the gel-liquid crystalline transition restored the currents. Translocation of R18 was also shown by fluorescence measurements. When R18 was in the bilayer at high, self-quenching concentrations, voltage pulses led to voltage-dependent fluorescence changes. The kinetics of the fluorescence changes and charge translocations correlated. Adding the quencher I- to one aqueous phase caused fluorescence to decrease or increase when voltage moved R18 toward or away from the quencher at low, nonquenching concentrations of R18. In contrast to R18, Dil incorporated into bilayers was a carrier fo I-, and hence I- altered Dil currents. Voltage-driven translocations allow R18 and Dil to be used to probe membrane potential changes.     

Membrane structure alterations in rat blood lymphocytes under external low dose rate gamma-radiation exposure

The influence of a 0.72 cGy/day dose rate of gamma-radiation on plasma membranes of peripheral blood lymphocytes of rats exposed to the doses of 1.5, 15, 30, 60 and 100 cGy was studied. Parameters characterizing the viscosity and the polarity of lipid bilayer and also an external membrane surface properties were examined using fluorescent probes pyrene and 1-anilinonaphthalene-8-sulfonate (ANS). Was shown the membrane structural parameters alterations after animal exposure to the doses of 1.5, 15, 60 and 100 cGy, being of a nonmonotonous nature as the dose accumulated. After exposure to the doses lower then than 30 cGy spectral changes were revealed not in each particular experiment that was probably caused by the individual peculiarities of radiation response development. After exposure to the doses higher than 30 cGy the changes were of reproducible character. After a 1.5 cGy dose a slight lipid bilayer polarity decrease and ANS binding parameter multidirectional changes were observed. After exposure to 15, 60 and to 100 cGy was shown polarity elevation and repartition of polar groups within the bilayer, the increase of viscosity of more polar membrane regions and also ANS fluorescence reduction mostly at the expense of quantum yield decrease. After the exposure of 60 cGy was observed a viscosity decrease in hydrophobic regions along with viscosity increase in more polar regions and after a 100 cGy dose accumulation an essential surface charge shift was found. Revealed alterations indicate the reorganization of external membrane surface and of intensification of oxidative processes in lipid bilayer.     

Environmental polarity estimation in living cells by use of quinoxaline-based full-colored solvatochromic fluorophore PQX and its derivatives

Fluorosolvatochromic probes have recently attracted interest for live cell imaging. We recently tested our quinoxaline-based fluorosolvatochromic probes, PQX and MPQX, for in vivo imaging applications. PQX and MPQX are characterized by donor (pyrrole site)-acceptor (quinoxaline site) structure, and the peak emission wavelength of these compounds varies over the visible wavelength range depending on the polarity of the solvent used, for a variety of solvents from hexane to water. A linear relationship was obtained between peak emission wavenumber and E(T)(N) (normalized solvent polarity). When tested on cultured HEp-2 cells, the results showed that the PQX and MPQX were not harmful at the applied concentrations (10(-5) M), and site-dependent fluorescence spectra in living cells were observed with PQX treatment, which indicates that PQX penetrates into the plasma membrane, followed by delocalization throughout the cells. MPQX was also able to penetrate the cell membrane, distribute throughout the cells and emit fluorescence, but did not show site-dependent intracellular fluorescence spectra. These results suggest that PQX is a remarkable tool for investigating the local polarity environment in cells after appropriate conjugation to biomolecules.     

Fluorescent membrane probes incorporating dipyrrometheneboron difluoride fluorophores.

The spectroscopic properties of a new series of fatty acid analogs in which a dipyrrometheneboron difluoride fluorophore forms a segment of the acyl methylene chain are presented and their characteristics as fluorescent membrane probes are examined. When incorporated as a low mole fraction component in model phospholipid membranes, the probes retain the principal characteristics of the parent fluorophore: green fluorescence emission with high quantum yield, extensive spectral overlap, and low environmental sensitivity. The fluorescence quantum yield is typically two to three times that of comparable membrane probes based on the nitrobenzoxadiazole fluorophore. The spectral overlap results in a calculated F?rster energy transfer radius (Ro) of about 57 A. Consequently, increasing fluorescence depolarization and quenching are observed as the mole fraction of the probe species incorporated in the membrane is increased. Low environmental sensitivity is manifested by retention of high quantum yield emission in aqueous dispersions of fatty acids. Partition coefficient data derived from fluorescence anisotropy measurements and iodide quenching experiments indicate that in the presence of fluid phase phospholipid bilayers the aqueous fraction of fatty acid is very small. Fluorescence intensity and anisotropy responses to phospholipid phase transitions are examined and found to be indicative of nonrandom fluorophore distribution in the gel phase. It is concluded that the spectroscopic properties of the fatty acid probes and their phospholipid derivatives are particularly suited to applications in fluorescence imaging of cellular lipid distribution and membrane level studies of lateral lipid segregation.     

Mg~(2+)加强嵌有H~+-ATP酶脂酶体脂质分子的堆积(packing)

用亲脂性的灵敏的荧光MC 540标记在有Mg~(2+)(1mM)与无Mg~(2+)条件下重建的线粒体H~+-ATP酶脂酶体,后者的荧光强度较前者增加30%左右.这提示,含Mg~(2+)的脂酶体的脂质分子间的堆积紧密度增加.在N-AF系列(n=27和16)探剂与MC 540之间的能量转移效率,又以反应靠近脂双层表面变化的2-AP与MC 54O之间最高.这进一步表明,含Mg~(2+)的脂酶体具有较适合流动性是与Mg~(2+)通过调节靠近脂双层表面的脂质分子具有适度的堆积相关的.这对阐明我们已提出的Mg~(2+)促进线粒体H~+-ATP酶重建作用模型进一步提供了较直接的证据.