Stimulated fluorescence and fluorescence quenching in chlorophyll a and bacteriochlorophyll a

This communication deals with the photophysical processes that take place in chlorophyll solutions under intense nitrogen laser irradiation. The effect of the pump photon density on the fluorescence yield depends strongly on the geometry of the irradiation and the sampling set-up. If the fluorescence cell and sampling probe are placed close to the transverse arrangement used for obtaining laser output, line narrowing and gain, which are processes associated with high population inversions and stimulated fluorescence, are observed. A normal fluorescence spectrum and a decrease in fluorescence quantum yield with increasing pump power are observed in the fluorescence cells in oriented at an angle of 20–40° with respect to the transverse axis of the exciting beam. The decrease in quantum yield appears to result from absorption of the pump photons by the excited singlet of the chlorophylls, and it is suggested that an analogous mechanism may be responsible for the anomalous fluorescence quantum yield reported for in in vivo Chlorella vulgaris algae.     

Photoinduced electron transfer of chlorophyll in lipid bilayer system

Photoinduced electron transfer from chlorophyll-a throughtheinterface of dipalmitoylphosphatidylcholine (DPPC) headgroup of the lipid bilayers was studied with electron magnetic resonance (EMR). The photoproduced radicals were identified with electron spin resonance (ESR) and radical yields of chlorophyll-a were determined by double integration ESR spectra. The formation of vesicles was identified by changes in measured λ_max values from diethyl ether solutions to vesicles solutions indirectly, and observed directly with SEM and TEM images. The efficiency of photosynthesis in model system was determined by measuring the amount of chlorophyll-a radical yields which were obtained from integration of ESRspectra.     

Simulations of lipid transfer between a supported lipid bilayer and adsorbing vesicles

Recent experiments demonstrate transfer of lipid molecules between a charged, supported lipid membrane (SLB) and vesicles of opposite charge when the latter adsorb on the SLB. A simple phenomenological bead model has been developed to simulate this process. Beads were defined to be of three types, ‘n’, ‘p’, and ‘0’, representing POPS (negatively charged), POEPC (positively charged), and POPC (neutral but zwitterionic) lipids, respectively. Phenomenological bead–bead interaction potentials and lipid transfer rate constants were used to account for the overall interaction and transfer kinetics. Using different bead mixtures in both the adsorbing vesicle and in the SLB (representing differently composed/charged vesicles and SLBs as in the reported experiments), we clarify under which circumstances a vesicle adsorbs to the SLB, and whether it, after lipid transfer and changed composition of the SLB and vesicle, desorbs back to the bulk again or not. With this model we can reproduce and provide a conceptual picture for the experimental findings.     

Partition and binding constants in micelles and vesicles from fluorescence quenching data

A method of analysing steady-state fluorescence quenching in compartmentalized systems is proposed which allows the evaluation of partition and binding constants where both association processes occur simultaneosly. The number of binding sites is shown to be dependent on the nature of the quencher.     

Tethered bilayer lipid micromembranes for single-channel recording: the role of adsorbed and partially fused lipid vesicles

A mercury-supported bilayer lipid micromembrane was prepared by anchoring a thiolipid monolayer to a mercury cap electrodeposited on a platinum microdisc about 20 μm in diameter; a lipid monolayer was then self-assembled on top of the thiolipid monolayer either by vesicle fusion or by spilling a few drops of a lipid solution in chloroform on the cap and allowing the solvent to evaporate. Single-channel recording following incorporation of the alamethicin channel-forming peptide exhibits quite different features, depending on the procedure followed to form the distal lipid monolayer. The "spilling" procedure, which avoids the formation of adsorbed or partially fused vesicles, yields very sharp single-channel currents lasting only one or two milliseconds. These are ascribed to ionic flux into the hydrophilic spacer moiety of the thiolipid. Conversely, the vesicle-fusion procedure yields much longer single-channel openings analogous to those obtained with conventional bilayer lipid membranes, albeit smaller. This difference in behavior is explained by ascribing the latter single-channel currents to ionic flux into vesicles adsorbed and/or partially fused onto the tethered lipid bilayer, via capacitive coupling.     

Cardanol as a replacement for cholesterol into the lipid bilayer of POPC liposomes

Large unilamellar liposomes were prepared by hydration of 1-palmitoyl-2-oleylphosphatydilcholine (POPC) films and subsequent extrusion of the obtained liposomal suspension. Inclusion of cholesterol and cardanol brings about a stabilization of the membranes of the liposomes, as determined by their rates of release of entrapped 5(6)-carboxyfluorescein. The liposome breakdown was promoted by a non-ionic surfactant (Triton X-100) and the kinetic measurements were carried out by fluorimetry in water at 25 degrees C. Morphological analyses of giant POPC liposomes in the presence and in the absence of both guests were also performed. The results obtained suggest the use of cardanol (an easy available natural product) as a replacement for cholesterol as a new possibility for stabilizing liposomes in drug targetting.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: detection by antibiotic membrane probes.

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylcholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmetrically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Preparation of highly photosensitizing liposomes with fullerene-doped lipid bilayer using dispersion-controllable molecular exchange reactions

Fullerene-containing liposomes with high photosensitization ability were prepared. Disaggregated fullerenes were efficiently injected into the bilayer of liposomes by a phototriggered molecular exchange reaction. These liposomes showed far higher photoreactivity than liposomes thermally produced by heating and microwave irradiation. This result indicates that control of self-aggregation of fullerene leads to a high quantum yield for the photoreaction because of the suppression of self-quenching of photoexcited fullerenes.     

Interaction of liposomes with silica nanocapsules: from lipid bilayer coating to multi-liposomal composites

A new delivery system has been developed that combines liposomes and silica nanocapsules. It was found that in the presence of cationic unilamellar liposomes, silica nanocapsules are instantly coated with a lipid bilayer. In contrast, cationic stealth liposomes modified with polyethylene glycol can adsorb on the surface of silica to form multi-liposomal composites in which the liposomes remain intact.     

Subnanometer actuation of a tethered lipid bilayer monitored with fluorescence resonance energy transfer

Lipid membrane nanotechnology can play a key role in preserving the function of transmembrane proteins on biofunctional substrates. We show here that rational nanoscopic actuation of a polymer-tethered lipid bilayer can be achieved by modulating the dielectric environment at the membrane-substrate interface. This provides a hydrated platform with increased lipid mobility compared to bilayers supported directly onto silica. We suggest that this construct may be used for promoting the functional reconstitution of transmembrane proteins on planar surfaces for bioanalytical devices.     

Magnetic quenching of fluorescence

The direct and indirect mechanisms for the magnetic enhancement of radiationless decay processes are described, and used to account for the experimental observations of the magnetic quenching of fluorescence from carbon disulphide, glyoxal, and nitrogen dioxide.     

The effects of a thiol-containing quinazolinedione derivative (MECH) on the lipid oxidation in bilayer liposomes

In order to investigate the radical chemistry of 3-(2-mercaptoethyl)quinazoline-2,4(1H, 3H)-dione (MECH) in homogeneous and liposomal solutions experiments were performed with pulse radiolysis, γ radiolysis and the chemical radical intiator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). It is shown that the thiol group represents the most sensitive group to radical attack. The thiyl radical originating from MECH is detected indirectly by product analysis and by pulse radiolysis. The thiyl radical can abstract bisallylic hydrogens from polyunsaturated fatty acids. This is shown by pulse radiolysis in homogeneous and liposomal solutions via the formation of the pentadienyl radical which has a strong and characteristic absorption band at 280 nm.     

The action of oxygen on chlorophyll fluorescence quenching and absorption spectra in pea thylakoid membranes under the steady-state conditions

The effect of oxygen concentration on both absorption and chlorophyll fluorescence spectra was investigated in isolated pea thylakoids at weak actinic light under the steady-state conditions. Upon the rise of oxygen concentration from anaerobiosis up to 412 microM a gradual absorbance increase around both 437 and 670 nm was observed, suggesting the disaggregation of LHCII and destacking of thylakoids. Simultaneously, an increase in oxygen concentration resulted in a decline in the Chl fluorescence at 680 nm to about 60% of the initial value. The plot of normalized Chl fluorescence quenching, F(-O(2))/F(+O(2)), showed discontinuity above 275 microM O(2), revealing two phases of quenching, at both lower and higher oxygen concentrations. The inhibition of photosystem II by DCMU or atrazine as well as that of cyt b(6)f by myxothiazol attenuated the oxygen-induced quenching events observed above 275 microM O(2), but did not modify the first phase of oxygen action. These data imply that the oxygen mediated Chl fluorescence quenching is partially independent on non-cyclic electron flow. The second phase of oxygen-induced decline in Chl fluorescence is diminished in thylakoids with poisoned PSII and cyt b(6)f activities and treated with rotenone or N-ethylmaleimide to inhibit NAD(P)H-plastoquinone dehydrogenase. The data suggest that under weak light and high oxygen concentration the Chl fluorescence quenching results from interactions between oxygen and PSI, cyt b(6)f and Ndh. On the contrary, inhibition of non-cyclic electron flow by antimycin A or uncoupling of thylakoids by carbonyl cyanide m-chlorophenyl hydrazone did not modify the steady-state oxygen effect on Chl fluorescence quenching. The addition of NADH protected thylakoids against oxygen-induced Chl fluorescence quenching, whereas in the presence of exogenic duroquinone the decrease in Chl fluorescence to one half of the initial level did not result from the oxygen effect, probably due to oxygen action as a weak electron acceptor from PQ pool and an insufficient non-photochemical quencher. The data indicate that mechanism of oxygen-induced Chl fluorescence quenching depends significantly on oxygen concentration and is related to both structural rearrangement of thylakoids and the direct oxygen reduction by photosynthetic complexes.     

Supramolecular chemistry in lipid bilayer membranes

Lipid bilayer membranes form compartments requisite for life. Interfacing supramolecular systems, including receptors, catalysts, signal transducers and ion transporters, enables the function of the membrane to be controlled in artificial and living cellular compartments. In this perspective, we take stock of the current state of the art of this rapidly expanding field, and discuss prospects for the future in both fundamental science and applications in biology and medicine.

This perspective provides an overview of the current state of the art in supramolecular chemistry in lipid bilayer membranes, including receptors, signal transducers, catalysts and transporters, and highlights prospects for the future.     

荧光猝灭法测定次黄嘌呤及机理研究

研究了次黄嘌呤猝灭伊文思蓝的荧光猝灭机理并根据次黄嘌呤对伊文思蓝荧光的猝灭作用建立了测定次黄嘌呤的新方法.线性回归方程为:ρ=6.292×103F-1-26.49,相关系数为0.9990,线性范围为0.20～20.0 μg/mL,检出限为0.14 μg/mL,相对标准偏差(RSD)为2.5%.求算了伊文思蓝与次黄嘌呤的形成常数K=2.63×102L/moL,实验了pH、放置时间、干扰离子等对测定的影响.     

Electronic processes and photosensitization in bilayer lipid membranes

Abstract— In part one of this paper, evidence for electronic processes in experimental and biological membranes are reviewed. The membrane under consideration, be it experimental or biological, is understood to mean an ultrathin bamer separating two aqueous phases. The question ‘can electronic processes occur in/across such a structure immersed in an aqueous environment?’ is answered affirmatively. In the second part of this paper, photosensitization by dyes and photoelectric effects in experimental bilayer lipid membranes observed recently are described.     

Thioamide quenching of intrinsic protein fluorescence

Thioamides quench tryptophan and tyrosine fluorescence in a distance-dependent manner and thus can be used to monitor the binding of thioamide-containing peptides to proteins. Since thioamide analogs of the natural amino acids can be synthetically incorporated into peptides, they can function as minimally-perturbing probes of protein/peptide interactions.     

Measurement of glucose using fluorescence quenching

A new spectroscopic procedure for the measurement of glucose concentrations is described which is based on substrate induced quenching (SIQ) of an indicator fluorescence. The method exploits a novel photo reaction between thionine and NADH, the latter being generated due to the reduction of NAD⁺ in an enzymic reaction between glucose dehydrogenase (GDH) and glucose. The observed SIQ data was analysed using an empirical relation. A quenching constant of 1.8×10³ (±100) M⁻¹ is obtained for the substrate induced quenching of thionine by glucose. The reported method, which was investigated over the range 0–1000 μM, offers a glucose detection limit of 2.2 μM. Various applications of the proposed scheme are discussed, including its use to construct a fibre optic biosensor for glucose.