Showing papers on "Fluorescence spectrometry published in 1991"

Poly(styrene-ethylene oxide) block copolymer micelle formation in water: a fluorescence probe study

Abstract: L'etude en fonction de la concentration du copolymere de la variation des proprietes spectroscopiques du pyrene provoquee par son partage entre les phases micellaires et aqueuses permet de determiner les concentrations critiques micellaires et les coefficients de partage

J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential.

Abstract: The spectral properties of a novel membrane potential sensitive probe (JC-1) were characterized in aqueous buffers and in isolated cardiac mitochondria. JC-1 is a carbocyanine with a delocalized positive charge. It formed under favorable conditions a concentration-dependent fluorescent nematic phase consisting of J-aggregates. When excited at 490 nm, the monomers exhibited an emission maximum at 527 nm and J-aggregates at 590 nm. Increasing concentrations of JC-1 above a certain concentration caused a linear rise in the J-aggregate fluorescence, while the monomer fluorescence remained constant. The membrane potential of energized mitochondria (negative inside) promoted a directional uptake of JC-1 into the matrix, also with subsequent formation of J-aggregates. The J-aggregate fluorescence was sensitive to transient membrane potential changes induced by ADP and to metabolic inhibitors of oxidative phosphorylation. The J-aggregate fluorescence was found to be pH independent within the physiological pH range of 7.15-8.0 and could be linearly calibrated with valinomycin-induced K+ diffusion potentials. The advantage of JC-1 over rhodamines and other carbocyanines is that its color altered reversibly from green to red with increasing membrane potentials. This can be exploited for imaging live mitochondria on the stage of a microscope.

The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen

Abstract: NHIK 3025 cells were incubated with Photofrin II (PII) and/or tetra (3-hydroxyphenyl)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i.e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 3-6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PII present in the same cells. The distance diffused by the reactive intermediate, supposedly mainly 1O2, causing the photodegradation was estimated to be on the order of 0.01-0.02 micron, which corresponds to a lifetime of 0.01-0.04 microsecond of the intermediate in the cells. PII has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PII under the present conditions. Photoproducts (1O2 etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.

Fluorescence ratio imaging of cyclic AMP in single cells

Abstract: Fluorescence imaging is perhaps the most powerful technique currently available for continuously observing the dynamic intracellular biochemistry of single living cells However, fluorescent indicator dyes have been available only for simple inorganic ions such as Ca2+, H+, Na+, K+, Mg2+ and Cl- We now report a fluorescent indicator for the adenosine 3',5'-cyclic monophosphate (cAMP) signalling pathway The sensor consists of cAMP-dependent protein kinase in which the catalytic (C) and regulatory (R) subunits are each labelled with a different fluorescent dye such as fluorescein or rhodamine capable of fluorescence resonance energy transfer in the holoenzyme complex R2C2 When cAMP molecules bind to the R subunits, the C subunits dissociate, thereby eliminating energy transfer The change in shape of the fluorescence emission spectrum allows cAMP concentrations and the activation of the kinase to be nondestructively visualized in single living cells microinjected with the labelled holoenzyme

Fluorescence assay for phospholipid membrane asymmetry.

Abstract: Highly fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl-lipid (NBD-lipid) analogues are widely used to examine lipid transport and membrane structure. We have developed a method for chemically modifying NBD-labeled lipids in both artificial and biological membranes. This was achieved by treating fluorescently labeled membranes with dithionite (S2O4(-2)). When small unilamellar vesicles containing NBD-labeled phospholipids were reacted with dithionite, only the fluorescent lipid located on the outer leaflet of the vesicles' bilayer was reduced. Seven different NBD-lipid analogues, including a fluorescent sterol, were reduced by treatment with dithionite to nonfluorescent 7-amino-2,1,3-benzoxadiazol-4-yl-lipid derivatives. To assess the feasibility of using this reagent in biological systems, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanol ami ne was inserted into the outer leaflet of the plasma membrane of CHO-K1 cells. Subsequent incubation of these cells with a nontoxic concentration of dithionite resulted in the complete loss of fluorescence from the plasma membrane. In contrast, when cells were permitted to endocytose some of their fluorescently labeled plasma membrane and then treated with dithionite, fluorescence at the plasma membrane was eliminated, while intracellular labeling was not affected. These data suggest that dithionite reacts with NBD-labeled lipids in the outer leaflet of membrane bilayers, producing nonfluorescent derivatives. We demonstrate how reduction of NBD-lipids with dithionite can be used to prepare asymmetrically labeled liposomes and to measure transverse-membrane asymmetry in vesicles. This method should be useful in many biochemical investigations, including the measurement of phospholipid translocase activity.

The protective function of the epidermal layer of rye seedlings against ultraviolet‐b radiation

Abstract: — Ultraviolet-B radiation induces the accumulation of UV-absorbing pigments of the flavonoid type in the epidermal layer of rye seedlings. The content of pigments identified as isovitexin derivatives was about 4 times higher in leaves preirradiated for up to 24 h with long wavelength UV-B radiation as compared to control plants without any UV-B pretreatment. Leaves preconditioned in this way could prevent or reduce damage to photosynthetic function caused by short wavelength UV-B radiation. Photosynthetic activity monitored by variable fluorescence was reduced in unprotected leaves compared to protected leaves when expressed as a fluorescence decrease ratio value, as quantum yield of photosystem II (Fvm/Fm) or as leaf vitality index (Fm/Fo).

Fluorescence studies of hydrophobically modified poly(N-isopropylacrylamides)

Abstract: Preparation et etude des proprietes en solution de copolymeres 100:1 et 200:1 du N-isopropyl acrylamide avec les N-decyl-, N-tetradecyl- et N-stearyl acrylamide et de copolymeres marques par un groupe pyrenyl. Les copolymeres du N-tetradecyl- et du N-stearylacrylamide forment des micelles au-dessous de la temperature critique LCST; au-dessus de la temperature LCST, les micelles sont detruites et les groupes hydrophobes sont distribues aleatoirement parmi les chaines agregees et effondrees de polymere

Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry.

Abstract: Information about the rheological characteristics of the aqueous cytoplasm can be provided by analysis of the rotational motion of small polar molecules introduced into the cell. To determine fluid-phase cytoplasmic viscosity in intact cells, a polarization microscope was constructed for measurement of picosecond anisotropy decay of fluorescent probes in the cell cytoplasm. We found that the rotational correlation time (tc) of the probes, 2,7-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein, and 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) provided a direct measure of fluid-phase cytoplasmic viscosity that was independent of probe binding. In quiescent Swiss 3T3 fibroblasts, tc values were 20-40% longer than those in water, indicating that the fluid-phase cytoplasm is only 1.2-1.4 times as viscous as water. The activation energy of fluid-phase cytoplasmic viscosity was 4 kcal/mol, which is similar to that of water. Fluid-phase cytoplasmic viscosity was altered by less than 10% upon addition of sucrose to decrease cell volume, cytochalasin B to disrupt cell cytoskeleton, and vasopressin to activate phospholipase C. Nucleoplasmic and peripheral cytoplasmic viscosities were not different. Our results establish a novel method to measure fluid-phase cytoplasmic viscosity, and indicate that fluid-phase cytoplasmic viscosity in fibroblasts is similar to that of free water.

Dynamics of mismatched base pairs in DNA.

Abstract: The structural dynamics of mismatched base pairs in duplex DNA have been studied by time-resolved fluorescence anisotropy decay measurements on a series of duplex oligodeoxynucleotides of the general type d[CGG(AP)GGC].d[GCCXCCG], where AP is the fluorescent adenine analogue 2-aminopurine and X = T, A, G, or C. The anisotropy decay is caused by internal rotations of AP within the duplex, which occur on the picosecond time scale, and by overall rotational diffusion of the duplex. The correlation time and angular range of internal rotation of AP vary among the series of AP.X mismatches, showing that the native DNA bases differ in their ability to influence the motion of AP. These differences are correlated with the strength of base-pairing interactions in the various AP.X mismatches. The interactions are strongest with X = T or C. The ability to discern differences in the strength of base-pairing interactions at a specific site in DNA by observing their effect on the dynamics of base motion is a novel aspect of the present study. The extent of AP stacking within the duplex is also determined in this study since it influences the excited-state quenching of AP. AP is thus shown to be extrahelical in the AP.G mismatch. The association state of the AP-containing oligodeoxynucleotide strand is determined from the temperature-dependent tumbling correlation time. An oligodeoxynucleotide triplex is formed with a particular base sequence in a pH-dependent manner.

Analysis of confocal laser-microscope optics for 3-D fluorescence correlation spectroscopy.

Abstract: Quantitative fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR) measurements in bulk solution require a well characterized confocal laser microscope optical system. The introduction of a characteristic function, the collection efficiency function (CEF), provides a quantitative theoretical analysis of this system, which yields an interpretation of the FCS and FPR measurements in three dimensions. We demonstrate that when the proper field diaphragm is introduced, the 3-D FCS measurements can be mimicked by a 2-D theory with only minor error. The FPR characteristic recovery time for diffusion is expected to be slightly longer than the corresponding time measured by FCS in the same conditions. This is because the profile of the laser beam used for photobleaching is not affected by the field diaphragm. The CEF is also important for quantitative analysis of standard scanning confocal microscopy when it is carried out using a finite detection pinhole.

Design of 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde as a reagent for ultrasensitive determination of primary amines by capillary electrophoresis using laser fluorescence detection

Abstract: Amino acids and peptides, from both standard solutions and biological samples, were successfully reacted with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde, at low concentration, to form highly fluorescent isoindole derivatives. The formed mixtures are effectively separated by high-performance capillary electrophoresis and their constituents detected by their laser-induced fluorescence signals. The minimum detectable quantities in the low attomole (10(-18) mol) range are encountered.

Fluorescence detection in capillary electrophoresis: evaluation of derivatizing reagents and techniques

Abstract: A capillary electrophoresis fluorescence detector is described. Detection is performed on-capillary with either deuterium, tungsten, or xenon arc lamps. A grating monochromator is used for the selection of the excitation wavelength. Optimized limits of detection using various sources are given for several analytes and compared to absorbance results and data from a breadboard laser-based system employing the same emission detector configuration. Separations are optimized for mixtures of amino acids labeled with various fluorescent reagents by using both pre- and postcapillary derivatizations

Propeptide of carboxypeptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity.

Abstract: The zymogen of the vacuolar carboxypeptidase Y from Saccharomyces cerevisiae was purified and characterized with respect to activation as well as refolding in vitro. The purified procarboxypeptidase Y has no detectable activity but can be efficiently activated by proteinase K from Tritirachium album. We used this method of activation as a tool for the investigation of refolding procarboxypeptidase Y in vitro. The proenzyme, denatured in 6 M guanidinium chloride, is renatured efficiently after dilution of the denaturant, whereas the mature enzyme regains little activity in the same procedure. Changes in intrinsic fluorescence reveal the mature enzyme to be considerably more stable than the proenzyme toward denaturation with guanidinium chloride. This suggests that the propeptide induces a metastable structure important for overcoming energy barriers that might otherwise obstruct a productive folding pathway. The relatively large number of charged amino acid residues and a high theoretical potential for alpha-helix formation in the carboxypeptidase Y propeptide suggest a structural similarity to a number of other propeptides and heat shock proteins.

Preparative, analytical and fluorescence spectroscopic studies of sulphonated aluminium phthalocyanine photosensitizers

Abstract: Fluorescence spectroscopic studies were carried out on aluminium phthalocyanine with defined numbers (mono, di, tri and tetra) of sulphonate groups. Selective sulphonation was achieved using one of two synthetic methods to prepare a mixture of components which were separated using reverse-phase liquid chromatography. Fluorescence lifetimes were measured in methanol and buffer solution using time-correlated single-photon counting with picosecond laser excitation; the lifetime shows little variation with the number of sulphonate groups. Using steady state excitation, fluorescence quantum yields were determined for the tetrasulphonated component (phi F = 0.51) and, for comparison, unsulphonated aluminium phthalocyanine.

Fluorescence detection in capillary zone electrophoresis using a charge-coupled device with time-delayed integration.

Abstract: A fluorescence detection system for capillary zone electrophoresis is described in which a charged-coupled device (CCD) views a 2-cm section of an axially illuminated capillary column. The CCD is operated in two readout modes: a snapshot mode that acquires a series of images in wavelength and capillary position, and a time-delayed integration mode that allows long exposure times of the moving analyte zones. By use of the latter mode, the ability to differentiate a species based on both its fluorescence emission and migration rate is demonstrated for fluorescein and sulforhodamine 101. The detection limit for fluorescein isothiocyanate (FITC) is 1.2 X 10(-20) mol; detection limits for FITC-amino acids are in the (2-8) X 10(-20) mol range.

Methods for measurement of intracellular magnesium: NMR and fluorescence.

Abstract: Accurate and reproducible methods for the measurement of cytosolic free magnesium ion concentration, Mgj, are required in order to assess its physiologic role. This review covers two methods, in vivo nuclear magnetic resonance (NMR) and fluorescence spectroscopy, which have provided useful means for the determin�ation of Mgj. The first section contains a general discussion of the extraction of Mgj values from NMR or fluorescence data for magnesium ion chelators. Subsequent sections cover the use of endogenous NMR magnesium chelators, exogenous NMR Mgj indicators, and fluorescent Mgj indicators. Two recent reviews (2, 15) covered Mgj determinations based on ATP measurements, but did not cQver more recently developed fluorinated or fluorescent indicators. Other methods are covered in References 2 and 34. With the availability of new Mgj indicators, our understanding of the role of magnesium in metabolic regulation and in the etiology of disease will un­ doubtedly increase in the near future.

Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives.

Abstract: Propidium has been demonstrated in previous studies to be a selective ligand for the peripheral anionic site on acetylcholinesterase (EC 3.1.1.7). Its association with this site can be advantageously monitored by direct fluorescent titration. We have measured the ability of acetylcholine, acetylthiocholine, haloxon [di-(2-chloroethyl)3-chloro-4-methylcoumarin-7-ylphosphate] , and a coumarin derivative (3-chloro-7-hydroxy-4-methylcoumarin) to dissociate propidium from the peripheral anionic site of Torpedo californica acetylcholinesterase. Measurements were made by back-titration of propidium after complete inhibition of the active center with diisopropylfluorophosphate. Both acetylcholine and acetylthiocholine show substrate inhibition at high substrate concentrations. The concentrations required for occupation of the peripheral site, as ascertained by competition with propidium, correlated well with the concentration dependence for the kinetics of substrate inhibition. These observations are consistent with substrate inhibition being due to binding of acetylcholine or acetylthiocholine at a peripheral anionic site. Displacement of propidium by haloxon and coumarin indicated that these inhibitors also bind to the peripheral anionic site. The dissociation constants ascertained from peripheral site occupation are in agreement with the constants obtained from inhibition kinetics. Evidence is presented that competition with propidium obtained by direct fluorescence titrations, when combined with inhibition kinetics, provides a more reliable means for ascertaining site selectivity of various inhibitors than does a kinetic analysis alone.

Cell-permeable fluorescent indicator for cytosolic chloride.

Abstract: A major limitation of quinolinium-based fluorescent indicators for cytosolic Cl- has been the necessity of invasive cell loading because the positively charged ring nitrogen confers high polarity and membrane impermeability A novel approach to mask the positive nitrogen was developed and evaluated for rapid, noninvasive indicator loading into living cells and effective intracellular trapping The nonpolar and lipophilic compound 6-methoxy-N-ethyl-1,2-dihydroquinoline (diH-MEQ) was Cl- insensitive but was readily oxidized to the membrane-impermeable and Cl(-)-sensitive fluorescent indicator 6-methoxy-N-ethylquinolium chloride (MEQ), MEQ had 344-nm absorbance and 440-nm emission maxima, 070 quantum yield, and 4100 M-1 cm-1 molar extinction coefficient In aqueous buffers, the fluorescence of MEQ was quenched by Cl- by a collisional mechanism with a Stern-Volmer constant (KCl) of 145 M-1 MEQ fluorescence was quenched by other anions (KBr = 275 M-1, KI = 360 M-1, KSCN = 300 M-1) but not by NO3-, SO4(2-), cations, and pH Swiss 3T3 fibroblasts and colonic T84 cells were loaded with MEQ by incubation at 37 degrees C with 25-50 microM diH-MEQ for 5-10 min followed by diH-MEQ-free buffer for 15 min MEQ stained cells brightly and uniformly and was nontoxic in studies of cell growth, cAMP and Ca2+ signaling, and electrophysiological properties MEQ leaked out of cells by less than 10% in 60 min and was sensitive to cytosolic Cl- with KCl = 19 M-1(ABSTRACT TRUNCATED AT 250 WORDS)

Role of intracellular-free calcium in the cornified envelope formation of keratinocytes: differences in the mode of action of extracellular calcium and 1,25 dihydroxyvitamin D3.

Abstract: Extracellular calcium (Cao) and the steroid hormone 1,25(OH)2D, induce the differentiation of human epidermal cells in culture. Recent studies suggest that increases in intracellular free calcium (Cai) levels may be an initial signal that triggers keratinocyte differentiation. In the present study, we evaluated cornified envelope formation, the terminal event during keratinocyte differentiation, and correlated it with changes in the Cai levels during differentiation of keratinocytes in culture induced by Cao or 1,25(OH)2D. Keratinocytes were grown in different Cao concentrations (0.1 or 1.2 mM) or in the presence of 1,25(OH)2D (10(-11) to 10(-7) M), and the Cai levels were measured using the fluorescent probe Indo-1. Our results suggest that the induction of cornified envelope formation is associated with an increase in Cai level during calcium-induced differentiation. Cao and the calcium ionophore ionomycin acutely increased Cai and cornified envelope formation. In contrast, the effect of 1,25(OH)2D on increasing Cai levels and stimulating cornified envelope formation was long-term, requiring days of treatment with 1,25(OH)2D. Our data are consistent with other recent studies and support the hypothesis that Cao regulates keratinocyte differentiation primarily by acutely increasing their Cai levels. The role of calcium in the mechanism of action of 1,25(OH)2D on keratinocyte differentiation is less clear. The increase in Cai of keratinocytes during 1,25(OH)2D induced differentiation may be essential for or subsequent to its prodifferentiation effects.