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Fluorescence Spectroscopy (fluorescence + spectroscopy)
Kinds of Fluorescence Spectroscopy Selected AbstractsFluorescence Spectroscopy of Color Centers Generated in Phosphate Glasses after Exposure to Femtosecond Laser PulsesJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 5 2002James W. Chan A confocal fluorescence microscopy setup was used to observe, in situ, spectral changes in phosphate glasses which were modified using 0.3 ,J of tightly focused 800 nm, 130 fs laser pulses. On 488 nm excitation, the modified glass shows a broad fluorescence centered at roughly 600 nm, which decays with prolonged exposure to the 488 nm light. The decay behavior is dependent on the 488 nm power, with a faster decay rate for higher powers. A mechanism whereby color centers, formed by the femtosecond pulses, fluoresce when excited by the 488 nm light and are simultaneously photobleached is proposed to explain the observed behavior. [source] Time-Resolved and Steady-State Fluorescence Spectroscopy of Eumelanin and Indolic PolymersPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2007Stephen P. Nighswander-Rempel Eumelanin plays a variety of important physiological roles in human skin. However, its structure and fundamental properties still remain poorly understood. Although the absorbance of eumelanin is broad and reveals little about its structure, a variety of techniques have revealed the presence of a disordered array of chromophores within the melanin compound. In order to examine the fluorescence decay dynamics of these chromophores, time-resolved spectroscopy was applied to solutions of synthetic eumelanin and a melanin-like polymer of N-methyl,5-hydroxy,6-methoxyindole (N-Me-5H6MI). Solutions were excited with 80 fs laser pulses at 355, 370, 390 and 400 nm, and decay time courses were acquired at 20 nm intervals between 400 and 600 nm for each excitation wavelength. Decay profiles for both eumelanin and the polymer exhibited a characteristic multiexponential behavior with decay times between 0.5 and 15 ns, although steady-state spectra for the polymer exhibited only two peaks. The long-decay component in the polymer showed a significant decrease in both amplitude (30,5%) and decay time (14,6 ns) with increasing emission wavelength. In contrast, the amplitude and decay time in melanin increased slightly (10,15% and 7,10 ns, respectively) from 400 to 520 nm emission, at which point they leveled off. These trends were consistent for all excitation wavelengths. These results suggest that the multiexponential behavior of melanin fluorescence is characteristic of each oligomer within the eumelanin compound, and is consistent with the assertion that the diversity of constituents within eumelanin provides it with a robustness in spectral properties. [source] Endogenous Fluorescence Spectroscopy of Cell Suspensions for Chemopreventive Drug Monitoring,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2005Nathaniel D. Kirkpatrick ABSTRACT Cancer chemopreventive agents such as N -4-(hydroxyphenyl)-retinamide (4HPR) are thought to prevent cancers by suppressing growth or inducing apoptosis in precancerous cells. Mechanisms by which these drugs affect cells are often not known, and the means to monitor their effects is not available. In this study endogenous fluorescence spectroscopy was used to measure metabolic changes in response to treatment with 4HPR in ovarian and bladder cancer cell lines. Fluorescence signals consistent with nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and tryptophan were measured to monitor cellular activity through redox status and protein content. Cells were treated with varying concentrations of 4HPR and measured in a stable environment with a sensitive fluorescence spectrometer. Results suggest that redox signal of all cells changed in a similar dose-dependant manner but started at different baseline levels. Redox signal changes depended primarily on changes consistent with NADH fluorescence, whereas the FAD fluorescence remained relatively constant. Similarly, tryptophan fluorescence decreased with increased drug treatment, suggesting a decrease in protein production. Given that each cell line has been shown to have a different apoptotic response to 4HPR, fluorescence redox values along with changes in tryptophan fluorescence may be a response as well as an endpoint marker for chemopreventive drugs. [source] Native Fluorescence Spectroscopy of Blood Plasma in the Characterization of Oral Malignancy,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2003S. Madhuri ABSTRACT Native fluorescence characteristics of blood plasma were studied in the visible spectral region, at two different excitation wavelengths, 405 and 420 nm, to discriminate patients with different stages of oral malignancy from healthy subjects. The fluorescence spectra of blood plasma of oral malignant subjects exhibit characteristic spectral differences with respect to normal subjects. Different ratios were calculated using the fluorescence intensity values at those emission wavelengths that give characteristic spectral features of each group of experimental subjects studied. These fluorescence intensity ratios were used as input variables for a multiple linear discriminant analysis across different groups. Leave-one out cross-validation was used to check the reliability of each discriminant analysis performed. The discriminant analysis performed across normal and oral cancerous subjects classified 94.7% of the original grouped cases and 93.7% of the cross-validated grouped cases. A classification algorithm was developed on the basis of the score of the discriminant functions (discriminant score) resulted in the analyses. The diagnostic potentiality of the present technique was also estimated in the discrimination of malignant subjects from normal and nonmalignant diseased subjects such as liver diseases. In the discriminant analysis performed across the three groups, normal, oral malignancy (including early and advanced stages) and liver diseases, 99% of the original grouped cases and 95.9% of the cross-validated grouped cases were correctly classified. Similar analysis performed across normal, early stage of oral malignancy, advanced oral malignancy and liver diseases correctly classified 94.9% of the original grouped cases and 91.8% of the cross-validated grouped cases. [source] Internalization of Aggregated Photosensitizers by Tumor Cells: Subcellular Time-resolved Fluorescence Spectroscopy on Derivatives of Pyropheophorbide-a Ethers and Chlorin e6 under Femtosecond One- and Two-photon Excitation,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2002L. Kelbauskas ABSTRACT Amphiphilic sensitizers self-associate in aqueous environments and form aggregated species that exhibit no or only negligible photodynamic activity. However, amphiphilic photosensitizers number among the most potent agents of photodynamic therapy. The processes by which these sensitizers are internalized into tumor cells have yet to be fully elucidated and thus remain the subject of debate. In this study the uptake of photosensitizer aggregates into tumor cells was examined directly using subcellular time-resolved fluorescence spectroscopy with a high temporal resolution (20,30 ps) and high sensitivity (time-correlated single-photon counting). The investigations were performed on selected sensitizers that exhibit short fluorescence decay times (<50 ps) in aggregated form. Derivatives of pyropheophorbide-a ether and chlorin e6 with varying lipophilicity were used for the study. The characteristic fluorescence decay times and spectroscopic features of the sensitizer aggregates measured in aqueous solution also could be observed in A431 human endothelial carcinoma cells administered with these photosensitizers. This shows that tumor cells can internalize sensitizers in aggregated form. Uptake of aggregates and their monomerization inside cells were demonstrated directly for the first time by means of fluorescence lifetime imaging with a high temporal resolution. Internalization of the aggregates seems to be endocytosis mediated. The degree of their monomerization in tumor cells is strongly influenced by the lipophilicity of the compounds. [source] Revealing the sfumato Technique of Leonardo da Vinci by X-Ray Fluorescence Spectroscopy,ANGEWANDTE CHEMIE, Issue 35 2010Laurence de, Viguerie Dr. Mehr als nur ein hübsches Gesicht: Nichtinvasive Röntgenfluoreszenzspektroskopie gibt eine Antwort auf die Frage, wie das Auftragen mehrerer Farbschichten übereinander mithilfe der sfumato -Technik funktioniert. Diese Technik wurde von Leonardo da,Vinci für die Gesichter in sieben seiner Bilder verwendet. Mit der neuen Methode lässt sich ganz klar eine große Vielfalt in da,Vincis Technik erkennen. [source] Phosphorus Ligand Imaging with Two-Photon Fluorescence Spectroscopy: Towards Rational Catalyst Immobilization,ANGEWANDTE CHEMIE, Issue 32 2010Fabrizio Marras Fleckenlose Katalysatoren: Die Ligandenimmobilisierung auf Trägern wurde durch Zweiphotonenfluoreszenzmikroskopie mit einem fluoreszierenden Nixantphos-Liganden als Sonde untersucht (siehe Bild). Im Immobilisierungsprozess bilden die Liganden Aggregate in Lösung und werden auf dem Träger abgeschieden, wo sie als helle Flecken im Fluoreszenzbild erscheinen. Indem man die Bildung der Aggregate verhindert, erhält man ,fleckenlose" Katalysatoren mit überlegenen Eigenschaften. [source] Cross-Reactive Sensor Arrays for the Detection of Peptides in Aqueous Solution by Fluorescence SpectroscopyCHEMISTRY - A EUROPEAN JOURNAL, Issue 1 2010Sébastien Rochat Abstract A simple but powerful method for the sensing of peptides in aqueous solution has been developed. The transition-metal complexes [PdCl2(en)], [{RhCl2Cp*}2], and [{RuCl2(p -cymene)}2] were combined with six different fluorescent dyes to build a cross-reactive sensor array. The fluorescence response of the individual sensor units was based on competitive complexation reactions between the peptide analytes and the fluorescent dyes. The collective response of the sensor array in a time-resolved fashion was used as an input for multivariate analyses. A sensor array comprised of only six metal,dye combinations was able to differentiate ten different dipeptides in buffered aqueous solution at a concentration of 50,,M. Furthermore, the cross-reactive sensor could be used to obtain information about the identity and the quantity of the pharmacologically interesting dipeptides carnosine and homocarnosine in a complex biological matrix, such as deproteinized human blood serum. The sensor array was also able to sense longer peptides, which was demonstrated by differentiating mixtures of the nonapeptide bradykinin and the decapeptide kallidin. [source] Study on the Interaction of Ketoconazole with Human and Bovine Serum Albumins by Fluorescence SpectroscopyCHINESE JOURNAL OF CHEMISTRY, Issue 12 2008Qing-Lian GUO Abstract The binding of ketoconazole to human serum albumin and bovine serum albumin was studied by using fluorescence and ultraviolet spectroscopy. The measurements were performed in 0.1 mol·L,1 phosphate buffer solution at pH=7.40±0.1. Decreasing of quenching constant was observed in association with temperature increase. Our findings show that the quenching mechanism of fluorescence of serum albumins by ketoconazole was static quenching because of compound formation. The thermodynamic parameters ,G, ,H, and ,S at different temperatures were calculated, showing that the electrostatic interactions and hydrophobic interaction are the main forces for the binding of ketoconazole to serum albumins. The distance r between the donor (Trp-214) and acceptor (ketoconazole) was obtained according to fluorescence resonance energy transfer theory. [source] Molecular Interaction between a Gadolinium,Polyoxometalate and Human Serum AlbuminEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 34 2009Li Zheng Abstract Polyoxometalates (POMs) show promising antibacterial, antiviral (particularly anti-HIV), antitumor, and anticancer activities, but the mechanism of these potential therapeutic effects remains to be elucidated at the molecular level. The interaction between the Gd-containing tungstosilicate [Gd(,2 -SiW11O39)2]13, and human serum albumin (HSA) was studied by several techniques. Fluorescence spectroscopy showed an energy transfer between the single tryptophan residue of HSA and the POM. Circular dichroism led to the conclusion that the POM significantly altered the secondary structure of HSA. Isothermal titration calorimetry revealed an enthalpy-driven binding reaction between HSA and the POM, resulting in the formation of a 1:1 complex.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] Biochemical characteristics of C-terminal region of recombinant chitinase from Bacillus licheniformis, implication of necessity for enzyme propertiesFEBS JOURNAL, Issue 9 2008Hsu-Han Chuang The functional and structural significance of the C-terminal region of Bacillus licheniformis chitinase was explored using C-terminal truncation mutagenesis. Comparative studies between full-length and truncated mutant molecules included initial rate kinetics, fluorescence and CD spectrometric properties, substrate binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, kcat/Km, was slightly increased for the truncated enzymes toward the soluble 4-methylumbelliferyl- N-N,-diacetyl chitobiose or 4-methylumbelliferyl- N - N,- N,-triacetyl chitotriose or insoluble ,-chitin substrate. By contrast, changes to substrate affinity, Km, and turnover rate, kcat, varied considerably for both types of chitin substrates between the full-length and truncated enzymes. Both truncated enzymes exhibited significantly higher thermostabilities than the full-length enzyme. The truncated mutants retained similar substrate-binding specificities and abilities against the insoluble substrate but only had approximately 75% of the hydrolyzing efficiency of the full-length chitinase molecule. Fluorescence spectroscopy indicated that both C-terminal deletion mutants retained an active folding conformation similar to the full-length enzyme. However, a CD melting unfolding study was able to distinguish between the full-length and truncated mutant molecules by the two phases of apparent transition temperatures in the mutants. These results indicate that up to 145 amino acid residues, including the putative C-terminal chitin-binding region and the fibronectin (III) motif of B. licheniformis chitinase, could be removed without causing a seriously aberrant change in structure and a dramatic decrease in insoluble chitin hydrolysis. The results of the present study provide evidence demonstrating that the binding and hydrolyzing of insoluble chitin substrate for B. licheniformis chitinase was not dependent solely on the putative C-terminal chitin-binding region and the fibronectin (III) motif. [source] Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cellsFEBS JOURNAL, Issue 4 2006Jorge Alegre-Cebollada Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS-7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose-dependent and time-dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent-resistant cellular membranes showed that sticholysin II colocalizes with caveolin-1 in fractions corresponding to raft-like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane-associated protein. Toxin binding to raft-like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS-7 cells through interaction with membrane domains which behave like cellular rafts. [source] Effect of Nonthermal Treatment on the Molecular Properties of Mushroom PolyphenoloxidaseJOURNAL OF FOOD SCIENCE, Issue 5 2003N.K. Sun ABSTRACT To elucidate the mechanism of inactivation of mushroom polyphenoloxidase (PPO) by nonthermal treatment, PPO solutions were irradiated up to 10 kGy or pressurized at 600 MPa and 800 MPa, respectively. PPO activities were significantly affected by , irradiation, and treatment at 5 kGy decreased the activity by 93%. Treatment of PPO at 600 MPa decreased the activity slightly, yet 10 and 20 min treatments at 800 MPa decreased the activities by 28% and 43%, respectively. Circular dichroism study showed that nonthermal treatment altered the ellipticity values in the range of 210 and 225 nm, resulting in decrease of the ordered structure. Fluorescence spectroscopy indicated that nonthermal treatment quenched the emission intensity. [source] Preparation, Characterization, and Photocatalytic Properties of CaNb2O6 NanoparticlesJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 2 2009In-Sun Cho CaNb2O6 nanoparticles with a size range of 30,50 nm were synthesized by heat treatment at 600°C after a solvothermal process and their optical and photocatalytic properties were investigated. The prepared powders were characterized by X-ray powder diffractometer, field-emission scanning electron microscope, transmission electron microscope, UV-Vis diffuse reflectance spectroscopy, Fluorescence spectroscopy, and Raman spectroscopy. Compared with a powder of the same material prepared by a solid-state reaction (SS) method, the nanoparticles exhibited a higher Brunauer,Emmett,Teller (BET) surface area, more efficient light absorption, and enhanced photocatalytic activity for producing H2 from pure water under UV irradiation. The photoluminescence spectra revealed that a radiative recombination process is dominant in the powder prepared by the SS method (strong blue emission at 300 K) under UV light irradiation, while no obvious emission was observed in the nanoparticles. This decrease of the radiative recombination as well as the higher optical absorption ability and higher BET surface area resulting from the reduced dimensionality led to enhanced photocatalytic activity of the nanoparticles. [source] Fluorescence Lifetime Spectroscopy of Glioblastoma Multiforme,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2004Laura Marcu ABSTRACT Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time-resolved, laser-induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time-resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time-resolved spectra were detected in a 360,550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral- (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high-grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectraland the time domain can enhance the ability of fluorescencebased techniques to diagnose and detect brain tumor margins intraoperatively. [source] Labeling of proteins with fluorescent probes: Photophysical characterization of dansylated bovine serum albumin,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 5 2003Valeria Levi Abstract Fluorescence spectroscopy is a widely used technique in biophysical studies. One of the strategies frequently used consists of labeling biomolecules with fluorescent probes, which have distinctive photophysical properties. This methodology allows the study of a wide variety of structural features of the biomolecule. We describe a simple laboratory activity for undergraduate Biophysical Chemistry courses. The experimental work includes two activities: labeling BSA with dansyl chloride and analyzing the resulting absorption and fluorescence spectra. The discussion of these activities helps students to understand the basis of fluorescence spectroscopy with emphasis in the application to biological systems. [source] Fluorescence spectroscopy of H-ras transfected murine fibroblasts: A comparison with Monte Carlo simulationsBIOPOLYMERS, Issue 2 2010Shlomo Mark Abstract Autofluorescence properties of tissues have been widely used to diagnose various types of malignancies. In this study, we measured the autofluorescence properties of H-ras transfected murine fibroblasts and the counterpart control cells. The pair of cells is genetically identical except for the transfected H-ras gene. We applied Monte Carlo simulations to evaluate the relative contributions of Rayleigh and Mie scattering effects towards fluorescence in an in vitro model system of normal and H-ras transfected fibroblasts. The experimental results showed that fluorescence emission intensity was higher for normal cells than the malignant counterpart cells by about 30%. In normal cells, linearity in emission intensity was observed for cell densities of up to 1.0 × 106 cells/ml whereas for transformed cells it was up to 1.4 × 106 cells/ml. Nuclear volume changes give good account for the differences in the intrinsic fluorescence between normal and malignant cells. The Monte Carlo (MC) code, newly developed for this study, explains both predominant experimental features: the large fluorescence intensity differences between the transfected and the corresponding control cells as well as the phenomena of the red shift in the excitation spectra as a function of cell density. The contribution of Rayleigh scattering was found to be predominant compared to Mie scattering. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 132,140, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] A spectroscopic investigation into the interactions of 3,- O -carboxy esters of thymidine with bovine serum albuminBIOPOLYMERS, Issue 9 2009Kalyan Sundar Ghosh Abstract Binding studies of 3,-O-carboxy esters of thymidine, reported inhibitors of ribonucleases, with bovine serum albumin (BSA) have been explored in this report. Fluorescence spectroscopy in combination with Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy have been used to determine the nature and mode of binding. The binding and quenching parameters were determined from tryptophan fluorescence quenching by Scatchard plots and modified Stern,Volmer plots. The association constants are of the order of 104 M,1 for both the ligands. Thermodynamic parameters suggest that apart from an initial hydrophobic association, hydrogen bonding and van der Waals interactions play a decisive role during protein-ligand complex formation. Minor changes were observed in the secondary structures of human serum albumin (HSA) as revealed by FTIR and CD. Docking studies suggest that the ligands are close to Trp 213, which causes fluorescence quenching. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 737,744, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Surface-enhanced Raman and steady fluorescence study of interaction between antitumoral drug 9-aminoacridine and trypsin-like protease related to metastasis processes, guanidinobenzoataseBIOPOLYMERS, Issue 2 2001Adrian Murza Abstract Fluorescence spectroscopy and surface-enhanced Raman spectroscopy (SERS) were applied to study the interaction of the antitumoral drug 9-aminoacridine (9AA) with a trypsin-like protease guanidinobenzoatase (GB) extracted from a mouse Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission was detected in the emission fluorescence spectra at certain drug and enzyme concentrations. A SERS study was accomplished on silver colloids at several excitation wavelengths in order to obtain more information about the interaction mechanism. The results derived from Raman spectroscopy indicated that 9AA in the amino monomeric form may interact with the enzyme by means of two different bonds: an ionic bond with a negatively charged amino acid and a ring stacking interaction with an aromatic residue placed in the catalytic site of GB. This interaction mechanism was responsible for a strong exciplex emission detected at a longer wavelength than the expected value of the normal fluorescence emission. Moreover, the GB concentration dependence of the interaction suggested that the drug was sensitive to the quaternary structure of the enzyme. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 85,94, 2001 [source] Regions of Tau Implicated in the Paired Helical Fragment Core as Defined by NMRCHEMBIOCHEM, Issue 10 2005Alain Sillen Dr. Abstract We have studied the mature Alzheimer-like fibers of tau by fluorescence and NMR spectroscopy. Assembly of the protein into paired helical filaments after incubation with heparin at 37,°C was verified by electron microscopy and size-exclusion chromatography. NMR spectroscopy on these mature fibers revealed different regions of residual mobility for tau: the N-terminal domain was found to maintain solution-like dynamics and was followed by a large domain of decreasing mobility; finally the core region was distinguished by a solid-like character. Heteronuclear-NOE data indicate that the decreasing mobility is due to both a slowing down of the rapid nanosecond movements and the introduction of slower movements that lead to exchange broadening. Fluorescence spectroscopy confirmed the presence of this rigid core, and some degree of protection from hydrogen exchange for those residues was observed. Hence, our data give a more precise picture of the dynamics of tau when it is integrated into mature filaments and should provide further understanding of the molecular processes that govern aggregation. [source] Novel Probes Showing Specific Fluorescence Enhancement on Binding to a Hexahistidine TagCHEMISTRY - A EUROPEAN JOURNAL, Issue 26 2008Mie Kamoto Abstract The introduction of hexahistidine (His tag) is widely used as a tool for affinity purification of recombinant proteins, since the His tag binds selectively to nickel,nitrilotriacetic acid (Ni2+,NTA) complex. To develop efficient "turn-on" fluorescent probes for His-tagged proteins, we adopted a fluorophore displacement strategy, that is, we designed probes in which a hydroxycoumarin fluorophore is joined via a linker to a metal,NTA moiety, with which it forms a weak intramolecular complex, thereby quenching the fluorescence. In the presence of a His tag, with which the metal,NTA moiety binds strongly, the fluorophore is displaced, which results in a dramatic enhancement of fluorescence. We synthesized a series of hydroxycoumarins that were modified by various linkers with NTA (NTAC ligands), and investigated the chemical and photophysical properties of the free ligands and their metal complexes. From the viewpoint of fluorescence quenching, Ni2+ and Co2+ were the best metals. Fluorescence spectroscopy revealed a 1:1 binding stoichiometry for the Ni2+ and Co2+ complexes of NTACs in pH,7.4 aqueous buffer. As anticipated, these complexes showed weak intrinsic fluorescence, but addition of a His-tagged peptide (H-(His)6 -Tyr-NH2; Tyr was included to allow convenient concentration measurement) in pH,7.4 aqueous buffer resulted in up to a 22-fold increase in the fluorescence quantum yield. We found that the Co2+ complexes showed superior properties. No fluorescence enhancement was seen in the presence of angiotensin,I, which contains two nonadjacent histidine residues; this suggests that the probes are selective for the polyhistidine peptide. [source] Synthesis, Structure, and Nonlinear Optical Properties of Cross-Conjugated Perphenylated iso -PolydiacetylenesCHEMISTRY - A EUROPEAN JOURNAL, Issue 1 2005Yuming Zhao Prof. Abstract Monodisperse, cross-conjugated perphenylated iso- polydiacetylene (iso- PDA) oligomers, ranging from monomer 15 to pentadecamer 25, have been synthesized by using a palladium-catalyzed cross-coupling protocol. Structural characteristics elucidated by X-ray crystallographic analysis demonstrate a non-planar backbone conformation for the oligomers due to the steric interactions between alkylidene phenyl groups. The electronic absorption spectra of the oligomers show a slight red-shift of the maximum absorption wavelength as the chain length increases from dimer 17,b to pentadecamer 25, a trend that has saturated by the stage of nonamer 22. Fluorescence spectroscopy confirms that the pendent phenyl groups present on the oligomer framework enhance emission, and the relative emission intensity consistently increases as a function of chain length n. The molecular third-order nonlinearities, ,, for this oligomer series have been measured via differential optical Kerr effect (DOKE) detection and show a superlinear increase as a function of the oligomer chain length n. Molecular modeling and spectroscopic studies suggest that iso- PDA oligomers (n>7) adopt a coiled, helical conformation in solution. [source] Chiral Induction, Memory, and Amplification in Porphyrin Homoaggregates Based on Electrostatic InteractionsCHEMPHYSCHEM, Issue 6 2009LiXi Zeng Dr. Abstract Supramolecular chirality in two configurational homoaggregates of anionic meso -tetrakis(4-sulfonatophenyl)porphyrin (TPPS) can be induced by D - and L -alanine in acidic water (see picture). The chirality can be further memorized and enforced through strong electrostatic interactions between TPPS aggregates and achiral poly(allylamine) [PAA]. Supramolecular chirality in two configurational homoaggregates of anionic meso -tetrakis(4-sulfonatophenyl)porphyrin (TPPS) can be induced by D - and L -alanine (Ala) in acidic water, respectively. The induced supramolecular chirality can be further memorized and enforced, even after complete removal of Ala or in the presence of excess Ala with the opposite configuration, through strong electrostatic interactions with achiral poly(allylamine) [PAA]. The ionic chiral interactions between TPPS and Ala or PAA are characterized by means of UV/Vis absorption and circular dichroism spectrometry. Fluorescence spectroscopy and atomic force microscopy are used as complementary techniques. On the basis of the comprehensive experimental results, a possible mechanism for chiral induction, memory, and amplification of TPPS homoaggregates by chiral amino acids and achiral PAA is proposed. Thus, we demonstrate a novel strategy to realize chiral memory in supramolecular systems by polyelectrolytes through hierarchical electrostatic self-assembly. [source] Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopiesFEBS JOURNAL, Issue 22 2003Tanya Sheynis Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid,water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides. [source] Nanoporous Alumina Membranes as Diffusion Controlling SystemsADVANCED FUNCTIONAL MATERIALS, Issue 12 2004S. Kipke Abstract This work describes the use of nanoporous alumina membranes for the diffusion of crystal violet molecules, encapsulated in the micelles of sodium dodecylsulfate (SDS), through pores ranging between 20 and 200,nm in diameter. The encapsulation of the crystal violet in SDS micelles is necessary in order to enlarge the size of the molecules to such an extent that the pore size becomes a speed-controlling function. Superior results were obtained when the membrane-containing capsule is placed into a water-filled beaker, and carefully moved by means of a "tipping bridge" in order to prevent diffusion problems in the capsule. Free crystal violet was liberated following diffusion due to the low SDS concentration in the aqueous solution, which was far below the critical micelle concentration (CMC). Micelle formation and encapsulation of crystal violet is shown by UV-visible and fluorescence spectroscopies. The experiments described herein serve as an exploratory test for developing novel drug delivery systems. [source] Anthrax vaccine powder formulations for nasal mucosal deliveryJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2006Ge Jiang Abstract Anthrax remains a serious threat worldwide as a bioterror agent. A second-generation anthrax vaccine currently under clinical evaluation consists of a recombinant Protective Antigen (rPA) of Bacillus anthracis. We have previously demonstrated that complete protection against inhalational anthrax can be achieved in a rabbit model, by intranasal delivery of a powder rPA formulation. Here we describe the preformulation and formulation development of such powder formulations. The physical stability of rPA was studied in solution as a function of pH and temperature using circular dichroism (CD), and UV-visible absorption and fluorescence spectroscopies. Extensive aggregation of rPA was observed at physiological temperatures. An empirical phase diagram, constructed using a combination of CD and fluorescence data, suggests that rPA is most thermally stable within the pH range of 6,8. To identify potential stabilizers, a library of GRAS excipients was screened using an aggregation sensitive turbidity assay, CD, and fluorescence. Based on these stability profiles, spray freeze-dried (SFD) formulations were prepared at pH 7,8 using trehalose as stabilizer and a CpG-containing oligonucleotide adjuvant. SFD formulations displayed substantial improvement in storage stability over liquid formulations. In combination with noninvasive intranasal delivery, such powder formulations may offer an attractive approach for mass biodefense immunization. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:80,96, 2006 [source] Conformational Effects on Photophysical Characteristics of C5,C5,-linked Dihydrothymine Dimers in Solution,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2000T. Ito ABSTRACT Photophysical characteristics of N-substituted C5,C5,-linked dihydrothymine dimers (1a,b[meso], meso compounds of [5R,5,S]-bi-5,6-dihydrothymines; 1a,b[rac], racemic compounds of [5R,5,R]-bi-5,6-dihydrothymines and [5S,5,S]-bi-5,6-dihydrothymines) in aqueous solution with varying contents of less-polar aprotic solvent such as tetrahydrofuran or dioxane have been investigated by UV-absorption, and steady-state and time-resolved fluorescence spectroscopies. Among the C5,C5,-linked dimers, (5R,5,S)-bi-5,6-dihydro-1-methylthymine (1a[meso]) showed a redshifted weak UV-absorption band at 270,350 nm and excimer fluorescence emission at ,max= 370 nm with a quantum yield (,F) of ,0.1 in phosphate buffer (pH < 10) at 293 K. Racemic compound of 5,6-dihydro-1-methylthymine dimer (1a[rac]), meso and racemic compounds of 5,6-dihydro-1,3-dimethylthymine dimers (1b[meso] and 1b[rac]) in phosphate buffer were nonfluorescent under similar conditions. The UV-absorption and fluorescence spectral characteristics of 1a[meso] in aqueous solution were interpreted in terms of intramolecular stacking interactions between the dihydropyrimidine chromophores leading to a preferential "closed-shell" conformation both in the ground state and the excited singlet state. In basic solutions at pH > pKa= 11.7, the fluorescence quantum yield of 1a[meso] decreased due to a dominant "open-shell" conformation resulting from the electrostatic repulsion between the deprotonated dihydrothymine chromophores of 1a[meso] in a dianion form. [source] Expression, purification, and analysis of unknown translation factors from Escherichia coli: A synthesis approachBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2010Justin D. Walter Abstract New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity of their unknown (from a list of potential candidates). Subsequent to the identification of their unknown translation factor, a series of protein unfolding exercises are performed employing circular dichroism and fluorescence spectroscopies, allowing students to directly calculate thermodynamic parameters centered around determining the equilibrium constant for unfolding as a function of denaturant (temperature or chemical). The conclusion of this multi-part laboratory exercise consists of both oral and written presentations, emphasizing synthesis of the roles of each translation factor during the stepwise process of translation. [source] Spectroscopic study on structure of horseradish peroxidase in water and dimethyl sulfoxide mixtureBIOPOLYMERS, Issue 2 2002Yasushi Maeda Abstract The structure of horseradish peroxidase (HRP) in phosphate buffered saline (PBS)/dimethyl sulfoxide (DMSO) mixed solvents at different compositions is investigated by IR, electronic absorption, and fluorescence spectroscopies. The fluorescence spectra and the amide I spectra of ferric HRP [HRP(Fe3+)] show that overall structural changes are relatively small up to 60% DMSO. Although the amide I band of HRP(Fe3+) shows a gradual change in the secondary structure and a decrease in the contents of , helices, its fluorescence spectra indicate that the distance between the heme and Trp173 is almost constant. In contrast, the changes in the positions of the Soret bands for resting HRP(Fe3+) and catalytic intermediates (compounds I and II) and the IR spectra at the CO stretching vibration mode of carbonyl ferrous HRP [HRP(Fe2+)-CO] show that the microenvironment in the distal heme pocket is altered, even with low DMSO contents. The large reduction of the catalytic activity of HRP even at low DMSO contents can be attributed to the structural transition in the distal heme pocket. In PBS/DMSO mixtures containing more than 70 vol % DMSO, HRP undergoes large structural changes, including a large loss of the secondary structure and a dissociation of the heme from the apoprotein. The presence of the components of the amide I band that can be assigned to strongly hydrogen bonding amide CO groups at 1616 and 1684 cm,1 suggests that the denatured HRP may aggregate through strong hydrogen bonds. © 2002 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 67: 107,112, 2002 [source] Novel Molecular Building Blocks Based on the Boradiazaindacene Chromophore: Applications in Fluorescent Metallosupramolecular Coordination PolymersCHEMISTRY - A EUROPEAN JOURNAL, Issue 15 2009Ö. Altan Bozdemir Dr. Abstract Bright polymers: Fluorescent coordination polymers made up of versatile functionalized bodipy (boron-dipyrrin) chromophore building blocks, such as that depicted, are described. Polymerization is signaled by changes in fluorescence emission intensity and shifts in peak emission wavelengths. We designed and synthesized novel boradiazaindacene (Bodipy) derivatives that are appropriately functionalized for metal-ion-mediated supramolecular polymerization. Thus, ligands for 2-terpyridyl-, 2,6-terpyridyl-, and bipyridyl-functionalized Bodipy dyes were synthesized through Sonogashira couplings. These fluorescent building blocks are responsive to metal ions in a stoichiometry-dependent manner. Octahedral coordinating metal ions such as ZnII result in polymerization at a stoichiometry corresponding to two terpyridyl ligands to one ZnII ion. However, at increased metal ion concentrations, the dynamic equilibria are re-established in such a way that the monomeric metal complex dominates. The position of equilibria can easily be monitored by 1H,NMR and fluorescence spectroscopies. As expected, although open-shell FeII ions form similar complex structures, these cations quench the fluorescence emission of all four functionalized Bodipy ligands. Bu çal,,mada, metal iyonlar, arac,l,,,yla supramoleküler polimerizasyon için uygun ,ekilde fonksiyonland,r,lm,, yeni boradiazaindasen (Bodipy) türevleri tasarlanm,, ve sentezlenmi,tir. Bu amaçla, ligand olarak Sonogashira reaksiyonu ile 2- ve 2,6-terpiridil ve bipiridil gruplar,n, içeren Bodipy boyarmaddeleri sentezlenmi,tir. Bu floresan yap, bloklar, stokiyometriye ba,l, bir biçimde metal iyonlar,na duyarl,l,k gösterirler. ZnIIgibi oktahedral koordinasyon e,ilimi olan metal iyonlar,, iki terpiridil ligand,na bir ZnIIiyonu tekabül edecek bir stokiyometride polimerizasyona yol açmaktad,rlar. Bununla beraber, yüksek metal iyonu deri,imlerinde monomerik metal kompleksinin bask,n olaca,, bir biçimde, dinamik dengeler yeniden kurulmaktad,r. Bu dengelerin pozisyonu1H,NMR ve fluoresans spektroskopileriyle kolayl,kla izlenebilmektedir. Beklenildi,i gibi, benzer kompleks yap,lar olu,turmas,na ra,men FeIIiyonu, sentezlenen tüm fonksiyonalize Bodipy ligandlar,n,n emisyonlar,n, sönümlendirmektedir. [source] | ||||||||||||||||||||||||||||||||||||||||