THE TRANSVERSE LOCATION OF FLUOROPHORES IN LIPID BILAYERS AND MICELLES AS DETERMINED BY FLUORESCENCE QUENCHING TECHNIQUES

Abstract— A fluorescence quenching technique has been used to determine the absolute partition coefficients of a set of n -doxyl stearates which partition between the aqueous and lipid phases of a phospholipid dispersion, and it is shown that the values of the coefficients vary in a systematic way depending on the position of the doxyl group on the acyl chain of the fatty acid. The n -doxyl stearates quench the fluorescence of n -(9-anthroyloxy) fatty acids in lipid bilayers, and consideration of the absolute partition coefficients of the quencher and of the microviscosity of the bilayer is necessary to extract information about the proximity of the fluorophore and quencher. In addition, quenching of the fluorescent fatty acids with a quencher which is small relative to the width of the lipid bilayer allows determination of both the absolute and local partition coefficients, the latter referring to a subvolume of the bilayer within which quenching occurs. The relationship between absolute and local partition coefficients is defined for micelles and bilayers and a method for determining the relative transverse positions of the anthroyloxy rings in these structures is described.     

Conjugated polyelectrolyte supported bead based assays for phospholipase A2 activity

A fluorescence based assay for human serum-derived phospholipase activity has been developed in which cationic conjugated polyelectrolytes are supported on silica microspheres. The polymer-coated beads are overcoated with an anionic phospholipid (1,2-dimyristoyl-sn-glycero-3-[phospho- rac-(1-glycerol)) (DMPG) to provide "lipobeads" that serve as a sensor for PLA2. The lipid serves a dual role as a substrate for PLA2 and an agent to attenuate quenching of the polymer fluorescence by the external electron transfer quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS). In this case quenching of the polymer fluorescence by AQS increases as the PLA2 digests the lipid. The lipid can also be used itself as a quencher and substrate by employing a small amount of energy transfer quencher substituted lipid in the DMPG. In this case the fluorescence of the polymer is quenched when the lipid layer is intact; as the enzyme digests the lipid, the fluorescence of the polymer is restored. The sensing of PLA2 activity has been studied both by monitoring fluorescence changes in a multiwell plate reader and by flow cytometry. The assay exhibits good sensitivity with EC50 values in the nanomolar range.     

Superquenching in cyanine pendant poly(L-lysine) dyes: dependence on molecular weight, solvent, and aggregation.

A series of poly(L-lysines) ranging in number of repeat units (N(PRU)) from 6 to 900 has been synthesized and the photophysics of the series and monomer cyanine dye have been studied in solution. In water or aqueous dimethyl sulfoxide, the oligomers and polymers exhibit high sensitivity to fluorescence quenching by oppositely charged electron acceptors; in this study, 9,10-anthraquinone-2,6-disulfonate was used as a quencher for the cationic fluorescent polyelectrolytes. Quenching constants (K(SV)) measured in 50:50 (v/v) dimethyl sulfoxide-water increase monotonically with increase in N(PRU) ranging from 630 M(-1) for monomer to 1.2 x 10(9) M(-1) for dilute solutions of the polymer having N(PRU) approximately 900. The polymers having N(PRU) > 100 exhibit predominantly J-aggregate absorption and fluorescence and enhanced susceptibility to quenching. For the polymers exhibiting strong J-aggregation, the effective exciton domain quenched by a single quencher reaches approximately 100 PRU. The results of this study permit a semiquantitative analysis of superquenching of fluorescent polyelectrolytes in solution and the factors that control it.     

Epoxy acids of the seed oil ofArtemisia absinthium

1. The seed oil of the Central Asian common wormwood has yielded 1.48% of cis-12,13-epoxyoctadec-cis-9-enoic acid, 5.94% of cis-9,10-epoxyoctadec-cis-12-enoic acid, and traces of 9,10-epoxyoctadecanoic acid, and their structures have been confirmed. 2. It is proposed to used the neutralization number of fatty acids to determine the amounts of epoxy acids in their combinations with α-hydroxydienic acids.     

13C NMR spectra of bromine derivatives of fatty acids

Summary The¹³C spectra of the methyl esters of the cis and trans isomers of 9,10-dibromostearic, of cis,cis-9,10,12,13-tetrabromostearic, and of 10,11-dibromoundecanoic acid have been obtained, and the assignment of all the signals has been performed. It has been shown that the presence of bromine atoms in the chain of a fatty acid leads to the magnetic non-equivalence of the¹³C nuclei of the methylene groups present at a distance of three carbon-carbon bonds from the nucleus to which the halogen is directly attached. A dependence has been found of the shift parameter of the carbon nuclei in a fatty acid chain on the relative arrangement of the bromine atoms.V. I. Lenin Tashkent State University. Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 26–30, January–February, 1977.     

New fluorescent chemosensors for silver ion

New fluorescent chemosensors, 1,8-bis(pyrazolylmethyl)anthracene and 9,10-bis(pyrazolylmethyl)anthracene, were synthesized. The 1,8-isomer showed selective fluorescent quenching effects with Ag(I) and Cu(II). On the other hand, the 9,10-isomer displayed a selective fluorescent quenching effect only with Ag(I). From the association constants obtained from fluorescent titrations and by extraction, we conclude that rigid immobilization of the ligands, 1,8-isomer, plays a more important role in the binding with Ag(I) than the additional pi-cation interaction offered by the 9,10-isomer.     

以二氰蒽为敏化剂的聚异戊二烯敏化光降解

<正> 近年来,以二氰蒽(DGA)为敏化剂对烯类化合物进行光敏氧化研究颇受重视。Foote等曾对此作过系统工作,他们从热力学、闪光光解以及对氧化产物进行分析等方法确认其间存在着电子转移。最近Steichen等指出:二氰蒽也可做为单线态氧敏化剂在光氧化反应中发挥作用。可以认为二氰蒽作为敏化剂兼有电子转移和能量转移的能     

Kinetic studies of the interaction of fatty acids with phosphatidylcholine vesicles (liposomes)

The kinetics of addition of fatty acids (as alkaline solutions of the fatty acid anions) to pre-existing unilamellar phospholipid vesicles (mean diameter 100 nm) has been studied. The phospholipid DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) has been mainly used, together with three fatty acids, oleic acid (cis-9-octadecenoic acid), linoleic acid (cis,cis-9,12-octadecadienoic acid) and capric acid (decanoic acid). Experiments were performed above as well as below the main phase transition temperature (Tm) of DMPC vesicles. The pH chosen to study the fatty acid vesicle interaction (after fatty acid and vesicle mixing) was 8.5 in the case of oleic acid and linoleic acid and 7.4 for capric acid. In the absence of any pre-existing phospholipid vesicles, the addition of alkaline solutions of the fatty acid anions to corresponding buffer solutions of pH 8.5 or 7.4 leads to a partial protonation of the fatty acid anions again resulting in the formation of fatty acid vesicles. This process is rather slow, taking place over a period of hours/days, and the vesicles formed are very polydisperse and include a range of vesicle sizes/shapes. However, in the presence of pre-existing phospholipid vesicles the added fatty acids equilibrate readily within a few minutes and the size of the vesicles that form are then closely related to the size of the originally present phospholipid vesicles; the vesicles formed being generally somewhat larger than the pre-existing vesicles. In the case of the phospholipid DMPC, the mixed fatty acid/phospholipid vesicle system is often formed rather rapidly (particularly above Tm), so that stopped-flow methods have been applied to follow the kinetics of the process. It is proposed that most of the fatty acid molecules are initially rapidly incorporated into the bilayers of the pre-exisiting phospholipid vesicles as monomers, rather than that the added fatty acids form separate fatty acid vesicles. The mean vesicle sizes formed in the systems investigated have been analysed by using dynamic light scattering measurements. The behaviour of the DMPC system was found to be slightly different from the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) system studied before, but the results are consistent with a model that involves growth and subsequent fission of the mixed vesicles. The study provides further support of the "matrix effect" in this type of system [S. Lonchin, P.L. Luisi, P. Walde, B.H. Robinson, J. Phys. Chem. B 103 (1999) 10910-10916]. The pre-existing DMPC vesicles act as a kind of seed to control the behavior of the system in the presence of added fatty acid anions.     

Quenching of fluorescence of 1-hydroxypyrene-3,6,8-trisulfonate (HPTS) by Cu2+, Co2+, Ni2+, I-, and cetylpyridinium (CP+) ions in water/AOT/heptane microemulsion

The quenching of the fluorescence of HPTS (1-hydroxypyrene-3,6,8-trisulfonate) by Cu(2+), Ni(2+), Co(2+), I(-), and CP(+) (cetylpyridinium cation) has been studied in the w/o microemulsion medium formed with water, AOT [sodium salt of bis (2-ethylhexyl) sulfosuccinic acid], and heptane as components at two [H(2)O]/[AOT] ratios (omega), 6 and 20. The quenching process has been found to be dynamic in nature. The lifetimes of HPTS in the microemulsion medium in the absence and in the presence of quencher have been determined. The analysis of the results has been performed in terms of the Stern-Volmer equation and the quenching sphere of action model. The Poisson distribution equation has been also used in the analysis of the probability of quencher distribution in the microemulsion compartment. The quenching of HPTS has been found to be much lower in microemulsion than in bulk water.     

Epoxy acids of the seed oil ofArtemisia absinthium

Summary 1. The seed oil of the Central Asian common wormwood has yielded 1.48% of cis-12,13-epoxyoctadec-cis-9-enoic acid, 5.94% of cis-9,10-epoxyoctadec-cis-12-enoic acid, and traces of 9,10-epoxyoctadecanoic acid, and their structures have been confirmed.2. It is proposed to used the neutralization number of fatty acids to determine the amounts of epoxy acids in their combinations with -hydroxydienic acids.3. The possibility has been shown of determining the amount of ratio of the isomeric epoxy acids using oxidative degradation of the total oxy acids.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 705–710, November–December, 1976.     

INCLUSION AND FLUORESCENE QUENCHING OF 2, 3-DIMETHYLNAPHTHALENE IN β-CYCLODEXTRIN CAVITIES

Abstract— The fluorescence quenching of 2,3-dimethylnaphthalene (DMN) incorporated to β-cyclodextrin (β-CD) cavities by different olefins (fumaronitrile, acrylonitrile, acrylamide and 2-hydroxyethylmethacrylate) has been measured as a function of the β-CD concentration. The quenching efficiency decreases when the β-CD concentration increases, but extrapolation of the data to infinite cavities concentration does not indicate complete protection. These results are interpreted in terms of two quenching processes, one of them taking place between 2,3-dimethylnaphthalene associated to a β-CD cavity and free quencher, and the other between the DMN and the quencher molecule, both associated with a different cavity. The rate constants of both quenching processes and the β-CD quencher association constant are obtained from the dependence of the quenching efficiency with β-CD concentration.     

A Photoswitchable Solvatochromic Dye for Probing Membrane Ordering by RESOLFT Super-resolution Microscopy**

A switchable solvatochromic fluorescent dyad can be used to map ordering of lipids in vesicle membranes at a resolution better than the diffraction limit. Combining a Nile Red fluorophore with a photochromic spironaphthoxazine quencher allows the fluorescence to be controlled using visible light, via photoswitching and FRET quenching. Synthetic lipid vesicles of varying composition were imaged with an average 2.5-fold resolution enhancement, compared to the confocal images. Ratiometric detection was used to probe the membrane polarity, and domains of different lipid ordering were distinguished within the same membrane.     

A fluorescence-based method for direct measurement of submicrosecond intramolecular contact formation in biopolymers: an exploratory study with polypeptides.

A fluorescent amino acid derivative (Fmoc-DBO) has been synthesized, which contains 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a small, hydrophilic fluorophore with an extremely long fluorescence lifetime (325 ns in H2O and 505 ns in D2O under air). Polypeptides containing both the DBO residue and an efficient fluorescence quencher allow the measurement of rate constants for intramolecular end-to-end contact formation. Bimolecular quenching experiments indicated that Trp, Cys, Met, and Tyr are efficient quenchers of DBO (k(q) = 20, 5.1, 4.5, and 3.6 x 10(8) M(-1) x s(-1) in D2O), while the other amino acids are inefficient. The quenching by Trp, which was selected as an intrinsic quencher, is presumed to involve exciplex-induced deactivation. Flexible, structureless polypeptides, Trp-(Gly-Ser)n-DBO-NH2, were prepared by standard solid-phase synthesis, and the rates of contact formation were measured through the intramolecular fluorescence quenching of DBO by Trp with time-correlated single-photon counting, laser flash photolysis, and steady-state fluorometry. Rate constants of 4.1, 6.8, 4.9, 3.1, 2.0, and 1.1 x 10(7) s(-1) for n = 0, 1, 2, 4, 6, and 10 were obtained. Noteworthy was the relatively slow quenching for the shortest peptide (n = 0). The kinetic data are in agreement with recent transient absorption studies of triplet probes for related peptides, but the rate constants are significantly larger. In contrast to the flexible structureless Gly-Ser polypeptides, the polyproline Trp-Pro4-DBO-NH2 showed insignificant fluorescence quenching, suggesting that a high polypeptide flexibility and the possibility of probe-quencher contact is essential to induce quenching. Advantages of the new fluorescence-based method for measuring contact formation rates in biopolymers include high accuracy, fast time range (100 ps-1 micros), and the possibility to perform measurements in water under air.     

Optical glucose detection across the visible spectrum using anionic fluorescent dyes and a viologen quencher in a two-component saccharide sensing system

A very general system is described in which anionic fluorescent dyes possessing a wide range of absorbance and emission wavelengths are used in combination with a boronic acid-modified viologen quencher to sense glucose at pH 7.4 in buffered aqueous solution. The present study demonstrates this capability with the use of eleven anionic fluorescent dyes of various structural types. Signal modulation occurs as the monosaccharide binds to the viologen quencher and alters its efficiency in quenching the fluorescence of the anionic dyes. The degree of quenching and the magnitude of the glucose signal were found to correlate roughly with the number of anionic groups on the dye. Optimal quencher : dye ratios were determined for each dye to provide a fairly linear signal in response to changes in glucose concentration across the physiological range.     

QUENCHING OF THE TRIPLET STATE OF CHLOROPHYLL a BY ASCORBIC ACID AND ASCORBATE IN ETHANOLIC SOLUTION

Abstract— Ascorbic acid and ascorbate in chlorophyll ethanol solution were found to be fairly efficient quenchers of the chlorophyll triplet state; comparable to the efficiency of ascorbic acid as a quencher in aqueous pyridine solution.
It has been well established that ascorbic acid quenches the triplet state of chlorophyll in aqueous pyridine solution.^(1,2) The bimolecular quenching constant, kQ , is very much less than that for O₂ or quinine.^(3,4)
Information regarding the quenching of the triplet state of chlorophyll by ascorbic acid in ethanolic solution is lacking. There has been some question as to whether ascorbic acid reduces photoexcited chloro-phyll-ethanolic solution because of its high oxidation potential, or because like the ascorbate ion, it acts only as a quencher; both ascorbic acid and ascorbate in high concentrations gave low quantum yields.⁽⁵⁾ The quenching of the triplet state by ascorbic acid and ascorbate was determined by the flash-photolytic method.     

Continuous glucose detection using boronic acid-substituted viologens in fluorescent hydrogels: linker effects and extension to fiber optics

A fluorescent anionic dye and a viologen appended with boronic acids, which serve as glucose receptors, have been synthesized and immobilized into a poly(2-hydroxyethyl methacrylate) hydrogel for use as a continuous glucose monitor. The fluorescence of the dye is modulated by the quenching efficiency of the viologen-based receptor, which in turn is dependent on the glucose concentration. Two monomeric versions of the quencher/receptor unit were prepared and their performance within the hydrogel evaluated. By tethering the quencher/receptor to the hydrogel matrix using a single-point attachment, slightly improved glucose sensing was observed. The hydrogels were tested for their ability to continuously and reversibly detect glucose over the course of several hours. The tests were carried out using a cuvette-based system, as well as a fiber-optic-based configuration. Under physiological conditions (0.1 M phosphate buffer, pH 7.4, 37 degrees C), the fluorescent hydrogels display an excellent dynamic response to glucose concentrations within the biologically significant range (2.5-20 mM).     

Anomalous association and fluorophore influence on the position of dimethylaniline in micelles: fluorescence quenching of 1,8-acridinedione

Fluorescence quenching of 9,10-dimethyl-3, 4,6,7,9,10-hexahydro-1,8(2H,5H) acridinedione (ADD) dye by N,N-dimethylaniline (DMA) in SDS and CTAB were studied by steady state fluorescence and time resolved techniques. The Stern-Volmer plots for the quenching of ADD by DMA is found to be linear and the Stern-Volmer constant K(SV) depends on the micellar concentration. The fluorescence quenching analysis reveals the binding of DMA with the micelles. The perturbation of the probe on the position of DMA molecule in micelle is inferred in the present investigation. The ADD fluorophore drives the DMA molecule into the non-polar region (core) of the micelle whereas other fluorophores like pyrene and rhodamine6G do not affect the position of DMA. In this report, the importance of the nature of fluorophores in determining the position and association of the quencher molecules in the aggregated systems is being discussed.     

FLUORESCENCE QUENCHING OF PYRENE DERIVATIVES BY NITROXIDES IN MICROHETEROGENEOUS SYSTEMS

Abstract Fluorescence quenching of pyrene derivatives by 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO), 4-oxo-2,2,6,6-tetramethylpiperidinyl-1-oxy and 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy has been measured in homogeneous solvents and microheterogeneous systems: cetyltrimethylammonium chloride micelles, large unilamellar vesicles of dioctadecyldimethylammonium chloride and dipalmitoylphosphatidylcholine (DPPC) and rat liver microsomes. The extent of intraaggregate quenching is mostly determined by the quencher incorporation to the micro-phases. In particular, it is observed that quenching by TEMPO in vesicles is considerably faster when the bilayer is in the liquid crystalline state. This significant increase in quenching rate with the melting of the bilayer is not observed for the other TEMPO derivatives, indicating that the effect of the lipid organization upon the solubility is related to the hydrophobicity of the solute. The data obtained in rat liver microsomes at 37°C show a pattern very similar to that observed in DPPC vesicles in the liquid crystalline state.     

Boronic acid-appended bis-viologens as a new family of viologen quenchers for glucose sensing

A new family of boronic acid-appended viologen quenchers has been synthesized containing two viologen subunits in a single quencher moiety. Relative to the single viologen-based quenchers previously developed in our laboratory, the bis-viologen ortho-boronic acid (BoB) compounds, in combination with the fluorescent dye, HPTS, display greatly enhanced Stern-Volmer quenching constants and much greater signal modulation in response to glucose. The superior performance is realized at lower quencher-to-dye ratios than are required for the single-viologen systems.     

Reactivity of singlet oxygen with tryptophan residues and with melittin in liposome systems

The reactivity of singlet oxygen, 1O2, with amino acids, polypeptides and proteins has been studied extensively in solution, in micelles and also in vesicles. Here we attempt to examine its reactivity with N-acetyltryptophan amide (NATA), with a tryptophan residue with a long aliphatic chain attached, Trp(CH2)16, and with melittin--a small membrane protein--in a solution containing liposomes. In such a heterogeneous system the sensitizer and/or the tryptophan residue can be located in the ambient D2O, in the liposome membrane or at the membrane-solution interface. The sensitizer meso-tetra(N-methyl-4 pyridyl)porphine tetratosylate (mTPTT) is located in the aqueous phase while hematoporphyrin (HP) is embedded in the membrane. The quenching of 1O2 by the tryptophan residues and by melittin in solution, when using either of the sensitizers, was compared with the data in the liposome-containing system. It was found that the location of the sensitizer and of the quencher in the liposome membrane or in the surrounding solution greatly affects the quenching rate constants of 1O2.