Fluorescent ligand

Studies on the neutrophil also benefit from the fact that considerable work has already gone into defining fluorescent ligands (formyl peptide agonists and antagonists and C5a) and real-time methodology for analyzing the dynamics of LRG interactions (3). 

Complexity of LR Dynamics in Intact Neutrophils at 37°. At present, our most versatile assay to analyze receptor binding uses fluorescent ligand and a high-affinity antibody to fluorescein which discriminates free from receptor-bound hexapeptide ligand (FLPEP, N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-Fl). The assay has a time resolution of 1... 

Real-time spectroscopic methods can be used to measure the binding, dissociation, and internalization of fluorescent ligands with cell-surface receptors on cells and membranes. The time resolution available in these methods is sufficient to permit a detailed analysis of complex processes involved in cell activation, particularly receptor-G protein dynamics. A description of the kinetics and thermodynamics of these processes will contribute to our understanding of the basis of stimulus potency and efficacy. 

Also recently, Liao and collaborators [89] proposed a homogeneous noncompetitive assay of a protein in biological samples based on FRET by using its tryptophan residues as intrinsic donors and its specific fluorescent ligand as the FRET acceptor, which was defined as an analytical FRET probe. To evaluate this method, a naphthylamine derivative, namely /V-biotinyl-/V -(l -naphthylj-ethylene-diamine (BNEDA) 33 was used as an analytical FRET probe for the homogeneous noncompetitive assay of streptavidin. 

Sugimoto T, Itagaki K, Irie K (2008) Design and physicochemical properties of new fluorescent ligands of protein kinase C isozymes focused on CH/7t interaction. Bioorg Med Chem 16 650-657... 

Liao F, Xie Y, Yang X et al (2009) Homogeneous noncompetitive assay of protein via Forster-resonance-energy-transfer with tryptophan residue(s) as intrinsic donor(s) and fluorescent ligand as acceptor. Biosens Bioelectron 25 112-117... 

Fluorescence energy transfer experiments, in which the energy transfer from the excited DNA bases to a fluorescent ligand is monitored by fluorescence excitation spectroscopy, has been used to analyze the binding of the bisquinolizinium species 35 to DNA <2004ARK219>. 

Fig. 1 (a) Chemical structures of fluorescent ligands metal ion coordination sites are indicated in blue, (b) Absorption (solid) and fluorescence spectra (dotted) of 1 (black) and its Zn2+ complex (red) in water at pH 7 whereas uncomplexed 1 is virtually nonfluorescent (fluorescence quantum yield of 1-Zn2+ amounts to 0.34 [31]... 

Fig. 2 Chemical structures of fluorescent ligands (anion coordination sites are indicated in blue) and tentative reaction schemes on interaction of 6 with chloride and fluoride in dichloromethane according to the color patterns observed in [41]. The wavelengths of absorption given in brackets have been calculated for the geometry optimized species 6, 6-C1 and 6 in the gas phase by semiempirical AMI calculations (Ampac V6.55, Semichem)...

Frigoli M, Ouadahi K, Larpent C (2009) A cascade FRET-mediated ratiometric sensor for Cu2+ ions based on dual fluorescent ligand-coated polymer nanoparticles. Chemistry 15 8319-30... 

Huang, X., Fluorescence polarization competition assay the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand, /. Biomol. Screen., 8, 34,2003. 

In some instances, flow cytometry assays are a superior alternative to conventional procedures for the determination of equilibrium binding constants (Stein et al., 2001). In contrast to assays that employ radiolabelled ligands, which measure population mean values for binding constants, flow cytometry methods can measure those values in individual cells, revealing heterogeneity in receptor expression within a population of cells or membrane vesicles. Furthermore, small samples can be characterized in a short period of time (hours). This approach to receptor-binding analysis may be limited only by the availability of a properly characterized fluorescent ligand. 

Wiley JS, Cebon JS, Jamieson GP, Szer J, Gibson J, et al. 1994. Assessment of proliferative responses to granulocyte-macrophage colony-stimulating factor (GM-CSF) in acute myeloid leukaemia using a fluorescent ligand for the nucleoside transporter. Leukemia 8 181-185. 

Fluorescence staining for flow cytometric analysis falls into three categories methods in which a fluorescent ligand accumulates on or within the cell (see Chapters 36,38) methods that require the ligand to interact with a cellular component to release the fluorophore or result in light emission (see Chapters 34,39) and methods that rely on fluorophore-coupled antibody binding (see Chapters 32,33). 

Fig. 3. If 1000-fold excess (10 M FMLPK) is added within 1 min after FMLPK-Fl exposure, the fluorescent ligand can be completely displaced from the receptors (- - ). Displacement of the FMLPK-Fl with 1000-fold FMLPK is less complete if neutrophils have been exposed to the fluorescent ligand for > 2 min.

An example of a continuously monitored reaction of 1 x 10 neutrophils before and after exposure to 1 x lO M is presented in Fig. 4. Most binding occurs within the first minute of fluorescent ligand exposure. A small, but detectable displacement of FMLPK-Fl is observed when 10-fold excess FMLPK is added to the reaction. 

Turcatti, G., Vogel, H., Chollet, A. Probing the binding domain of the NK2 receptor with fluorescent ligands evidence that heptapeptide agonists and antagonists bind differently. Biochemistry 1995, 34, 3972-3980. 

Fluorescence Spectrum Fluorescence spectroscopy Tryptophan environment, presence of fluorescent ligands or impurities.b... 

On the other hand, Koike et al. [3] described a fluorescent ligand which was selective for zinc(II) ions. The photoactive part of the molecule was in this case the dansyl moiety, connected to a cyclene unit by a diethylamino linker. In order to shift the analytical range to higher concentrations, a similar structure containing [12]aneN3 instead of cyclene was also prepared (Scheme 4). 

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