Coumarin Fluorophore Research Articles

A Fluorescent Receptor Assay for Benzodiazepines Using Coumarin-Labeled Desethylflumazenil as Ligand

This article describes a novel nonisotopic receptor assay for benzodiazepines with fluorescence detection. As labeled ligand (coumarin-labeled desethylflumazenil, CLDEF), a metabolite of the benzodiazepine antagonist flumazenil (desetheylflumazenil, Ro15-3890) has been coupled to a coumarin fluorophore, via a spacer. CLDEF had a Ki of 6.5 nM. To avoid the interference of the background fluorescence of the receptors in the measurement step, the bound CLDEF was dissociated from the receptors after the filtration step. This dissociation was achieved by incubating the CLDEF-bound to the receptors on the filters-with a weakly acetate buffer. The second filtrates then contained the previously bound CLDEF, which was then quantitated with a RP-HPLC system with a fluorescence detector. The results with a fluorescent receptor assay were very similar to those with a radioreceptor assay, in that the IC50 values of lorazepam were 7.2 +/- 0.5 and 6.6 +/- 0.7 nM, respectively.

Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli.

The mechanism of Pi interaction with phosphate binding protein of Escherichia coli has been investigated using the A197C mutant protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC-PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after binding, concomitant with a conformational change of the protein that closes the cleft containing the Pi binding site. Cleft closure has a rate constant of 317 s-1 (pH 7.0, 5 degrees C), and opening has a rate constant of 4.5 s-1. The fluorescence increase is likely to arise from a change in the hydrophobic environment during this closure as the steady state fluorescence emission (lambdamax and intensity) on Pi binding is mimicked by the addition of ethanol to aqueous solutions of an MDCC-thiol adduct. Fluorescence lifetimes in the absence and presence of Pi were 0.3 and 2.4 ns, respectively, consistent with the change in quantum yield. The rotational correlation time of the coumarin increases only 2-fold from 15 to 26 ns on binding Pi as measured by time-resolved polarization, consistent with the main rotation being determined by the protein even in the open conformation, but with greater local motion. Circular dichroism of the coumarin induced by the protein is weak in the absence of Pi and increases strongly upon saturation by Pi. These data are also consistent with an open to closed conformational model.

Crystal structure of phosphate binding protein labeled with a coumarin fluorophore, a probe for inorganic phosphate.

Crystal structures are presented for the A197C mutant of Escherichia coli phosphate binding protein (PBP) and the same mutant labeled at Cys197 with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). Both proteins are complexed with inorganic phosphate. The latter molecule, MDCC-PBP, exhibits a large increase in fluorescence on binding inorganic phosphate. The resulting high-fluorescence state of the coumarin and the ability of this coumarin to monitor the conformational changes associated with inorganic phosphate binding are interpreted in terms of the specific interactions of MDCC with the protein. The structure helps to explain why this particular label gives a high-fluorescence state on binding inorganic phosphate, while several other related labels do not, and hence aids our general understanding of environmentally sensitive fluorescence probes on proteins.

The ATPase activity in isometric and shortening skeletal muscle fibres.

Muscle proteins utilise the hydrolysis of ATP to provide the energy for force development and the production of mechanical work. We have developed a technique with high sensitivity and time resolution to probe as directly as possible the link between ATPase activity, force development and muscle shortening. The ATPase activity was recorded in real time during contraction and shortening of permeabilised muscle fibres of rabbit skeletal muscle by measuring fluorescence changes associated with the binding of inorganic phosphate, a product of ATPase activity, to a genetically engineered phosphate binding protein labelled with a coumarin fluorophore. The muscle shortening velocity was found to affect directly the ATPase activity, with up to a five-fold increase during shortening at moderate velocities, and a decrease in activity during slow stretch.

Synthesis and binding properties of 2H‐1‐benzopyran‐2 one fluorophore‐linked calcium channel antagonist nifedipine (1,4‐dihydropyridine) analogs

AbstractNifedipine analogs I, II and III with the 2H‐1‐benzopyran‐2‐one (coumarin) fluorophore linked at the 2, 2 and 6 and 4 positions of the dihydropyridine ring were synthesized by the modified Hantzch condensation procedures. Attempts to synthesize dihydropyridine with the fluorophore at position 3 (IV) were unsuccessful.

Three-photon excitation of 2,5-bis(4-biphenyl)oxazole: steady-state and time-resolved intensities and anisotropies

Three-photon excitation of 2,5-bis(4-biphenyl) oxazole (BBO) was observed when it was excited with the fundamental output of a femtosecond Ti:sapphire laser above 820 nm. The emission spectrum of BBO was identical for one-, two-, and three-photon excitation at 320, 640, and 960 nm, respectively. In toluene and triacetin, the emission intensity of BBO depended on the square of the laser power for wavelengths below 820 nm and displayed a sharp transition to a cubic dependence at longer wavelengths. The spatial distribution of the emission of BBO with three-photon excitation was more strongly localized than for two-photon excitation of a coumarin fluorophore at the same wavelength. The same single exponential intensity decay was observed for one-, two-, and three-photon excitation. However, the frequency domain anisotropy decay with threephoton excitation at 960 nm revealed a larger time-zero anisotropy, larger differential polarized phase angle, and larger modulated anisotropy than is possible for two-photon excitation with colinear oscillators. In triacetin, the anisotropy is not constant for three-photon excitation at different wavelengths. Surprisingly, the fluorescence intensities for three-photon excitation were only about 100-fold less than for two-photon excitation. The increasing availability of Ti:sapphire lasers suggests that multiphoton excitation can become a common tool in fluorescence spectroscopy.

Crowned-coumarin as a New Fluorescence Derivatization Reagent for Carboxylic Acids

Abstract 3-Bromoacetyl-6,7-[2′,3′-(1′,4′,7′,10′,13′-pentaoxacyclopentadeca)-2′-ene]coumarin (2) was prepared as a new fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatographic analysis. The derivatization of lauric acid with 2 was readily accomplished under a quite mild condition to yield a highly fluorescent lauric acid derivative. The reaction was accelarated by combining crown moiety as a catalytic site to coumarin fluorophore.

A new way of biosensing using fluorescence energy transfer and Langmuir-Blodgett films

A method of detecting the binding of ligand molecules (mannosides) to a biological receptor (Con A) by Förster energy transfer from Langmuir-Blodgett (LB) films is presented. The LB films consist of pre-polymerized amphiphilic polymethacrylates containing a coumarin fluorophore (donor) and reactive groups to which biological receptors can be attached. Studies determining the stability of the system are described. The specific binding of rhodamine (acceptor)-labelled ligands and their competitive replacement by unlablled analytes is demonstrated. A simple dual-channel fluorescence detector optimized for this purpose is described as well.

A novel photoactivatable cross-linker for the functionally-directed region-specific fluorescent labeling of proteins.

A cleavable cross-linking reagent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3'- dithiopropionate (SAED), was synthesized for the selective transfer of a coumarin fluorophore from a 'donor' protein to a position near the binding site of an interacting 'target' protein. SAED contains a terminal N-sulfosuccinimidyl ester for conjugation to the donor, a terminal photoactivatable azido-coumarin species for cross-linking with the interacting target, and a central disulfide spacer for the release of the labeled target after cleavage. To evaluate the effectiveness of this labeling reagent, soybean trypsin inhibitor (STI) was derivatized (approximately 0.5 mol/mol) with SAED and then photolyzed in the presence of trypsin. A single fluorescent cross-linked species (6-7 mol% of total STI) was observed by SDS/PAGE and, after reductive cleavage, was shown to be a 1:1 STI-trypsin complex. This complex was not detected without photolysis or with an inactivated cross-linker. Importantly, complex formation was inhibited by an excess of unmodified STI and prevented by substitution of a non-interacting protein for trypsin. Cleavage of the cross-linked complex revealed that the trypsin, but not the STI, was fluorescent; the uncomplexed trypsin fraction remained unlabeled. These results demonstrated the specificity of the labeling of trypsin by fluorescent-transfer cross-linking with SAED. An efficiency of about 15% for this cross-linking mediated labeling of trypsin was calculated. The short cross-linking span of SAED (less than or equal to 1.8 nm) strictly limited the labeling to the vicinity of the contact region of trypsin with STI. Thus, this novel cross-linker permits the region-specific targeting of a fluorophore near a functionally important binding site.

Substantivity of Various Anionic Surfactants Applied to Wool

Four compounds from each of the sodium alkyl sulphate, 3-alkyl-7-hydroxy cou marin sulphonate, and alkaryl-2-pyrazoline sulphonate series of anionic surfactants were applied to wool under conditions such that equilibrium distribution would occur throughout the fibers. The treated wools were then extracted under various conditions of time, pH, and temperature, and also with various surfactants or alcohols in the extraction solutions. Aqueous extractions, even at pH 10, were relatively ineffective in removing the more surface active members of each series. Solutions containing alcohols, especially isopropanol/pH 10 buffer (1:1), were particularly effective in ex tracting the surfactants from wool. The substantivities to wool of fluorescent anionic surfactants containing coumarin or pyrazoline fluorophores are similar to those of the sodium alkyl sulphate surfactants of similar critical micelle concentration.