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At least 253 records · Page 14Linked to original sources
PubMed2001

Single-molecule detection technologies in miniaturized high throughput screening: binding assays for g protein-coupled receptors using fluorescence intensity distribution analysis and fluorescence anisotropy.

G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based binding assays that are highly amenable to miniaturization. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those induced by binding of fluorescent ligands to membrane particles with multiple receptor sites. As a confocal detection technology, FIDA inherently allows reduction of the assay volume to the microliter range and below without any loss of signal. Binding and displacement experiments are demonstrated for various types of GPCRs, such as chemokine, peptide hormone, or small-molecule ligand receptors, demonstrating the broad applicability of this method. The results correlate quantitatively with radioligand binding data. We compare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. While FA requires a higher degree of binding, FIDA is sensitive down to lower levels of receptor expression. Both methods are, within these boundary conditions, applicable to uHTS.

Drug Evaluation, Preclinical↗
PubMed2004

Utilization of fluorescence polarization and time resolved fluorescence resonance energy transfer assay formats for SAR studies: Src kinase as a model system.

High-throughput screening (HTS), a major component of lead identification, often utilizes fluorescence-based assay technologies. For example, HTS kinase assays are formatted using a variety of fluorescence-based assay technologies including, but not limited to, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), time-resolved fluorescence resonance energy transfer (TR-FRET), and fluorescence polarization (FP). These assays offer tremendous advantages such as a nonradioactive format, ease of automation, and excellent reproducibility. Fluorescence-based assays frequently used for lead identification can also be useful for structure activity relationship (SAR) studies during lead optimization. An important issue when assessing an assay to be used for SAR is the ability of the assay to discriminate high-affinity small molecule inhibitors (pM-nM) from low-affinity inhibitors (microM-mM). The purpose of this study was to utilize HTS-friendly assay formats for SAR by developing TR-FRET, FP, and DELFIAassays measuring Src kinase activity and to define the theoretical lower limit of small molecule inhibitor detection achievable with these assay formats. The authors show that 2 homogeneous assay formats, TR-FRET and FP, allowed for the development of Src kinase assays with a lower limit of detection of K(i) = 0.01 nM. This study indicates that assay technologies typically used for HTS can be used during lead optimization by providing quantitative measurements of compound activity critical to driving SAR studies.

Biological Assay↗
PubMed2001

Binding of agonist but not antagonist leads to fluorescence resonance energy transfer between intrinsically fluorescent gonadotropin-releasing hormone receptors.

We have used spot fluorescence photobleaching recovery methods to measure the lateral diffusion of GnRH receptor (GnRHR) fused at its C terminus to green fluorescent protein (GFP) after binding of either GnRH agonists or antagonist. Before ligand binding, GnRHR-GFP exhibited fast rates of lateral diffusion (D = 18 +/- 2.8 x 10(-10)cm2 x sec(-1)) and high values for fractional fluorescence recovery (%R) after photobleaching (73 +/- 1%). Increasing concentrations of agonists, GnRH or D-Ala6-GnRH, caused a dose-dependent slowing of receptor lateral diffusion as well as a decreased fraction of mobile receptors. Increasing concentrations of the GnRH antagonist Antide slowed the rate of receptor diffusion but had no effect on the fraction of mobile receptors, which remained high. To determine whether the decrease in %R caused by GnRH agonists was due, in part, to increased receptor self-association, we measured the fluorescence resonance energy transfer efficiency between GnRHR-GFP and yellow fluorescent protein-GNRHR: There was no energy transfer between GnRHR on untreated cells. Treatment of cells with GnRH agonists led to a concentration-dependent increase in the energy transfer between GnRH receptors to a maximum value of 16 +/- 1%. There was no significant energy transfer between GnRH receptors on cells treated with Antide, even at a concentration of 100 nM. These data provide direct evidence that, before binding of ligand, GnRHR exists as an isolated receptor and that binding of GnRH agonists, but not antagonist, leads to formation of large complexes that exhibit slow diffusion and contain receptors that are self-associated.

Animals↗
PubMed2000

The enhanced green fluorescent protein as a tool for the analysis of protein dynamics and localization: local fluorescence study at the single-molecule level.

The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein-protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe-64-->Leu, Ser-65-->Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single-protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher-intensity emission after blue-light excitation with respect to wild-type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged-protein dynamics and fluorescence-activated cell sorting, but not for monitoring single-protein trafficking over extended periods of time.

Base Sequence↗
PubMed2003

Single-molecule fluorescence lifetime and anisotropy measurements of the red fluorescent protein, DsRed, in solution.

Fluorescence lifetime and anisotropy measurements were made on the red fluorescent protein (DsRed) from tropical coral of the Discosoma genus, both at single-molecule and bulk concentrations. As expected from previous work, the fluorescence lifetime of DsRed in solution is dependent on laser power, decreasing from an average fluorescence lifetime in the beam of about 3.3 ns at low power (3.5 ns if one extrapolates to zero power) to about 2.1 ns at 28 kW/cm2. At the single-molecule level, exciting with 532 nm, 10 ps laser pulses at 80 MHz repetition rate, DsRed particles entering the laser beam initially have a lifetime of about 3.6 ns and convert to a form having a lifetime of about 3.0 ns with a quantum yield of photoconversion on the order of 10(-3) (calculated in terms of photons per DsRed tetramer). The particles then undergo additional photoconversion with a quantum yield of roughly 10(-5), generating a form with an average lifetime of 1.6 ns. These results may be explained by rapid photoconversion of one DsRed monomer in a tetramer, which acts as an energy transfer sink, resulting in a lower quantum yield for photoconversion of subsequent monomers. Multiparameter correlation and selective averaging can be used to identify DsRed in a mixture of fluorophores, in part exploiting the fact that fluorescent lifetime of DsRed changes as a function of excitation intensity.

Fluorescence Polarization↗
PubMed1998

[Synthesis of new pyrazoline fluorescent compounds and their IR spectra and fluorescence property].

The Fluorescent compounds of pyrazoline have been used in many field. According to Schellhammer theory, we designed and synthesized five new benzothiazoyl pyrazoline compounds. Their infrared spectra and fluorescent spectra of five compounds were detected. The results showed that five compounds have fluorescence, fluorescence emission wavelength is about 450nm. Effect of different substituents on the fluorescent intensity was investigated.

Fluorescent Dyes↗
PubMed2004

Use of chimeric fluorescent proteins and fluorescence resonance energy transfer to monitor cellular responses.

In recent years, the development of new technologies based on the green fluorescent protein and fluorescence resonance energy transfer has introduced a new perspective in the study of cell biology. Real-time imaging of fluorescent biosensors has made it possible to directly visualize individual molecular events as they happen in intact, live cells, providing important and original insights for our understanding of biologically relevant problems. This review discusses some essential methodological aspects concerning the generation and use of fluorescence resonance energy transfer-based biosensors and presents selected examples of specific applications that highlight the power of this technology.

Animals↗
PubMedDate not supplied

Capillary electrophoresis of the collagen crosslinks HP and LP utilizing absorbance, wavelength-resolved laser-induced fluorescence and conventional fluorescence detection.

A capillary electrophoretic (CE) method is presented for the determination of the collagen crosslinks hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP). Various detection techniques are compared, i.e. UV-Vis diode-array absorbance detection (DAD) and fluorescence detection both in the laser-induced fluorescence (LIF) and the conventional fluorescence mode. LIF detection was performed using a frequency-doubled Rhodamine dye laser pumped by an excimer laser, for excitation at 290 and 325 nm. The emission was measured with an intensified diode-array detector mounted on a spectrograph to obtain wavelength-resolved spectra. Relevant concentration detection limits were achieved only by using LIF detection, i.e. 200 nM of HP and LP in a 30 mM phosphate buffer (pH 2.0). Linear calibration curves were obtained from the detection limits up to the maximum concentration available, 23 microM for HP and 4.2 microM for LP, respectively for both fluorescence modes. The identity of the migrating compounds was confirmed by on-line recording of both the absorption and the fluorescence spectra.

Buffers↗
PubMed2001

Allophycocyanin 1 as a near-infrared fluorescent tracer: isolation, characterization, chemical modification, and use in a homogeneous fluorescence resonance energy transfer system.

Allophycocyanin 1 (APC1), isolated from Mastigocladus laminosus, retains the same (alpha-beta)(3) trimeric structure as allophycocyanin (APC), but incorporates a peptide linker in its core leading to a 28% increase in its fluorescence quantum yield compared to APC. Moreover, APC1 exhibits an unexpectedly good stability at very low concentrations, at extreme pHs, or diluted in a low ionic strength medium whereas, under the same conditions, APC dissociates into an (alpha-beta) monomer, indicating that the peptide linker acts as a stabilizer of its trimeric structure. APC1 crosslinking experiments performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide gave a high reaction yield (95%) and showed a similar crosslinking process as previously described for APC. Fluorescence quantum yields of crosslinked APC1 or APC decrease by 20% after labeling on antibody or streptavidin. However, quantum yields of the crosslinked APC1 conjugates remain 25% higher than those of crosslinked APC conjugates. Associated with a europium trisbipyridine cryptate as donor, crosslinked APC1 was compared with crosslinked APC as acceptor in homogeneous time resolved fluorescence technology based on a fluorescence resonance energy transfer process. Using crosslinked APC1, assay performances were increased by 20%, showing that APC1 could be considered as a very promising near infrared fluorescent probe to replace APC in its biological applications.

Chromatography, Gel↗
PubMed1980

Specific fluorescent bands on chromosomes produced by acridine orange after prestaining with base specific non-fluorescent DNA ligands.

Metaphase chromosomes stained with acridine orange exhibit uniform yellow-green fluorescence. Chromosome preparations treated with the non-fluorescent A-T specific antibiotic distamycinA prior to acridine orange staining exhibit longitudinal fluorescent banding patterns similar to those produced by a number of fluorescent R-band techniques. Similarly, chromosome preparations treated with the non-fluorescent G-C specific antibiotic actinomycin D followed by acridine orange staining exhibit "Hoechst-type" banding patterns. Interactions of various ligand-DNA combinations in solution indicate that the base pair specific antibiotics induce banding patterns by selectively altering acridine orange binding sites in chromosomal regions rich in the particular base pair for which the antibiotic exhibits specificity.

Acridine Orange↗
PubMed2006

Creation of circularly permutated yellow fluorescent proteins using fluorescence screening and a tandem fusion template.

By experimenting with many different circularly permutated yellow fluorescent protein (cpYFP) variants as acceptors in fluorescence resonance energy transfer based biosensors, the optimal dynamic range can be discovered by sampling the possibilities of relative fluorophore orientations before and after bioactivity. Hence, to facilitate the sampling process, we introduced a new approach to construct a library of cpYFP variants using fluorescence screening and a tandem fusion template. This new approach is rapid because it does not require creating intermediate N- and C-terminal fragments and it allows quick screening for positive colonies by fluorescence. As a demonstration, eleven cpYFP variants were created and eight showed fluorescence. The emission and excitation spectra of these cpYFP variants showed strong similarity to YFP and therefore can be used in replacement.

Bacterial Proteins↗
PubMed1991

Fluorescence properties of angiotensin II analogues in receptor-simulating environments: relationship between tyrosinate fluorescence lifetime and biological activity.

Nanosecond time-resolved decays from excited-state tyrosinate fluorescence of angiotensin II analogues were measured from the emission at 350 nm. Fluorescence lifetimes were determined in several different solvents using N-acetyltyrosinamide as the reference standard. Long-lifetime tyrosinate fluorescence (LTF) of angiotensin II (ANG II) was observed in propylene glycol, trifluoroethanol and isopropanol but not in DMSO or water. The addition of SDS at a concentration sufficient to induce micelle formation in water resulted in LTF for ANG II. LTF for ANG II was longer in propylene glycol (21 ns) than in isopropanol (16 ns) whereas the % conformer(s) producing LTF was higher in isopropanol (79%) than in propylene glycol (19%). For a series of ten angiotensin analogues, LTF values determined in propylene glycol and isopropanol were a reflection of the contractile activities of these analogues in the rat uterus assay. In propylene glycol, with the notable exception of ANG III, biologically active analogues had longer LTFs (13-21 ns) than inactive analogues (0-11 ns). In isopropanol, strong agonists had longer LTFs (13-16 ns) than weak agonists/inactive analogues (0-11 ns). Structure-fluorescence relationships suggest that the primary TyrOH acceptor in ANG II is the His6 imidazole group, and that the C-terminal carboxylate has an essential auxiliary role in generating long-lived tyrosinate fluorescence. The present findings appear to support the proposition that the receptor conformation of ANG II contains a tripartite interaction of Tyr, His and carboxylate groups which is analogous to that found at the active site of serine proteinases, and that the tyrosinate nucleophile may activate angiotensin receptors. Solvents of intermediate polarity such as propylene glycol and isopropanol appear to induce conformations for small peptides such as angiotensin which resemble those present at membrane receptors.

Angiotensin II↗
PubMed1995

Dispersion state of phospholipids and fluorescence production with peroxidation in organic solvents: investigated by time-resolved fluorescence technique.

Fluorescent substances were found to be produced efficiently when phospholipids containing phosphatidylethanolamine (PE) and linoleic chains were autoxidized in non-polar solvents. By using these fluorescent substances as intrinsic probes, the dispersion state of phospholipids was investigated in various organic solvents. Fluorescence anisotropy decay measurements indicated that the aggregation size of phospholipids was much larger in hexane than in chloroform, methanol and tert-butyl alcohol. The average diameter of phospholipid aggregates in hexane was calculated to be 4-6 nm, which was dependent on the lipid composition. A consistent result was obtained when N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-gly cer o-3- phosphoethanolamine (NBD-PE) was used as an extrinsic probe. Comparison of the fluorescence data with small-angle X-ray scattering (SAXS) data suggested that a reverse micellar structure of phospholipids formed in hexane. It was shown that phospholipid aggregation enhanced the extent of peroxidation as well as the production yield of fluorescent substances of phospholipid.

Chromatography, Thin Layer↗
PubMed1977

The influence of temperature and urea on intrinsic fluorescence of Streptomyces subtilisin inhibitor. A study by fluorescence polarization and quenching.

Intrinsic fluorescence of new microbial protease inhibitor, Streptomyces subtilisin inhibitor was studied by observing fluorescence polarization degree and lifetime in the temperature range 25-81 degrees C. Striking thermal changes in these fluorescence properties of tryptophan residues were observed. The apparent molecular volumes for tryptophan and tyrosine residues in the native form were determined to be 89 and 75 A3, respectively. The fluorescence quenching by Br- or Cs+ was investigated to obtain a microenvironmental information around tryptophan residues both in the native and denatured form. Cs+ quenches the fluorescence slightly stronger than Br-, implying that there is not any distinctive electrostatic interaction between tryptophan residues and their neighborhood.

Bacterial Proteins↗
PubMed1981

Characterization of fluorescent 2-dimethylaminonaphthalene-5-sulfonyl-immunoglobulin G conjugates for application in fluorescence polarization studies.

Preparations of normal rabbit IgG antibody (NgG) and purified rabbit IgG antibody of anti-2,4-dinitrophenyl (Anti-Dnp) specificity were covalently labeled with the fluorescent probe 2-dimethylaminonaphthalene-5-sulfonyl chloride (2,5-Dns-Cl). 2,5-Dns-immunoglobulin G preparations were shown to have average fluorescence lifetimes of 29--32 ns by cross-correlated phase and modulation fluorescence lifetime measurements. Excitation maxima were observed at 275 and 368 nm, while the emission maximum occurred at 465 nm. With 390 nm excitation, an intrinsic fluorescence polarization of 0.43 was observed. Molar extinction coefficients of 36 000 M-1 . cm-1 (at 258 nm) and 2090 M-1 . cm-1 (at 352 nm) were determined by synthesis of 2,5-Dns-L-[4,5-3H]leucine, a tritiated sulfonamide of 2,5-Dns. Low labelling (1 mol 2,5-Dns/mol of antibody) of rabbit Anti-Dnp IgG antibody with 2,5-Dns-Cl did not significantly alter the antibody association constant with Dnp. Fluorescence polarization measurements were accurately measured (0.3% error) at antibody concentrations greater than 10(-7) M, using photon counting instrumentation. Rotational relaxation times of 128 and 131 ns were determined for 2,5-Dns-NgG and 2,5-Dns-Anti-Dnp, respectively.

Animals↗
PubMed2005

Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance.

The beta-galactosidase protein generated by the bacterial LacZ gene is widely used to map gene expression patterns. The ease of its use is only rivaled by green fluorescent protein, which can be used in combination with various other procedures such as immunocytochemistry, flow cytometry, or tract tracing. The beta-galactosidase enzymatic reaction potentially provides a more sensitive assay of gene expression than green fluorescent protein. However, the virtual impermeability and tendency to absorb light over a wide range limit the use of the most frequently used beta-galactosidase substrate, X-Gal, in combination with other fluorescent labeling procedures. Here, we provide details on a simple photoactivation procedure that transforms the light-absorbing X-Gal product, 5-bromo-4-chloro-3-indolyl (BCI) precipitate, into an intensely fluorescent product excited by 488 and 633 nm light. Photoactivation is achieved through exposure to 730 nm near-infrared light emitted from a femtosecond titanium-doped Sapphire laser. Photoactivation of BCI occurs in tissue sections suspended in buffered saline, glycerol, or even embedded in epoxy resin. A protocol for the use of BCI photoactivation is here provided. Importantly, the BCI photoactivated product is photoswitchable, displaying bistable photochromism. This permits the use of the fluorescent product in a variety of co-localization studies in conjunction with other imaging modalities. As with other bistable and photoswitchable products, the BCI reaction product shows concentration quenching at high density and can be degraded by continuous exposure to intense 730 nm illumination. Therefore, care must be taken in developing imaging strategies. Our findings have implications for the use of X-Gal in gene and protein detection and provide a novel substrate for high density digital information storage.

Animals↗
PubMed2005

Fluorescence quenching and time-resolved fluorescence studies on Trichosanthes dioica seed lectin.

Fluorescence quenching and time-resolved fluorescence studies have been carried out on the Trichosanthes dioica seed lectin (TDSL). The emission lambdamax of native TDSL, seen at 328nm, shifts to 343nm upon denaturation with 6M guanidinium chloride. Quenching titrations were performed with neutral (acrylamide and succinimide) and ionic (I(-) and Cs(+)) quenchers in order to probe the exposure and accessibility of tryptophan residues of the protein. Maximum quenching was observed with acrylamide, followed by succinimide, iodide and Cs(+). Dramatic increase in the extent of quenching and other quenching parameters by all the quenchers were observed upon denaturation of TDSL, suggesting that all the tryptophan residues in native TDSL are buried in the hydrophobic core of the protein. Increase in the extent of quenching upon denaturation of TDSL was maximum with I(-) and minimum with Cs(+), suggesting the presence of positively charged residue(s), near at least one tryptophan residue. Addition of saccharide ligands such as methyl-beta-d-galactopyranoside and lactose led to a small, but reproducible decrease in the fluorescence intensity of the lectin. The presence of lactose provided a partial protection against quenching by I(-), Cs(+) and succinimide, but not acrylamide. In time-resolved fluorescence measurements the fluorescence decay curves could be best fitted to biexponential patterns with lifetimes of 4.09 and 1.53ns for native lectin, 3.40 and 1.65ns for the lectin in presence of 0.1M lactose and 3.50 and 1.40ns for denatured lectin.

Fluorescence↗
PubMed1998

The effect of photodegradation on the fluorescent properties of norfloxacin: (photodegradation and fluorescence of norfloxacin).

Norfloxacin (NFLX), a broad spectrum antibacterial quinolone, is a very thermostable but photosensitive drug, especially in solution leading to the formation of an ethylenediamine degradate. The modification of the fluorescent properties of NFLX in acid solution after exposure to fluorescent light and the degradation mechanisms were studied. Two analytical methods were previously developed and validated for NFLX, ultraviolet spectrophotometry (UV) and spectrofluorimetry (FL). Data obtained using both methods in the analysis of remaining NFLX in terms of percent recoveries revealed that there was no statistically significant modifications of the UV signal and of the recoveries obtained by the method. However, an important increase of the fluorescent signal after light exposure of NFLX solutions appeared, which led to an increase of the average recovery up to 270% over 15 months. Using a previously validated HPLC method for the photostability studies of NFLX, a loss of 5% with respect to the initial drug amount was observed. The study of UV and fluorescence spectra evidenced the formation of the degradation product, which induced significant modification of the fluorescent properties of NFLX samples. These results clearly indicated that FL analysis definitively is the method of choice and can be used to study the photodegradation of NFLX.

Anti-Infective Agents↗