Fluorescent chelating indicators

Intracellular calcium elevation is monitored by fluorescent chelators developed by Tsien and coworkers. These indicators are loaded into cells the same way the pH indicators are. With Quin-2 (14), one of the first such probes developed, the quantum yield increases about fourfold when Ca binds to it. The second generation of Ca probes, Indo-1 and Fura-2 (15), are now being widely used in a variety of cell types. These probes are in most cases... 

Class 2 fluorophores that can reversibly bind an analyte. If the analyte is a proton, the term fluorescent pH indicator is often used. If the analyte is an ion, the term fluorescent chelating agent is appropriate. Fluorescence can be either quenched upon binding (CEQ type Chelation Enhancement of Quenching), or enhanced (CEF type Chelation Enhancement of Fluorescence). In the latter case, the compound is said to be fluorogenic [e.g. 8-hydroxyquinoline (oxine)]. 

Most of the fluorescent calcium indicators and their cell-permeant acetoxymethyl (AM) esters are variations of the nonfluorescent calcium chelator BAPTA and have been proposed by Tsien/134-1365 Among them Fura-2 and Indo-1 (Figure 5.22) are particularly used formeasuring Ca2+in single cells by imaging or flow cytometry/65... 

Fluorescent indicators for ions are based on corresponding chelators. The indicators usually mimic the structure of ethyleneglycol- / 5(P-aminoethylether)-A, A/,/V, /V -tetraacetic acid (EGTA), which also contains an adjacent fluorophore moiety affected by the binding. The most popular intracellular Ca " indicator, Fura-2, displays a shift in excitation maximum between 300-400 nm when monitoring emission at 510 nm, thus allowing ratiometric measurements. Calcium Green displays an increase in emission at 530 nm on addition of calcium but displays no spectral shift. Other fluorescent calcium indicators with different affinities and spectral characteristics are available that allow for the measurement of from nano- to submillimolar concentrations. 

Takeuchi and Yamamoto have developed a D-glucuronic acid selective fluorescent system (33) based on a boronic add and metal chelate [90, 91). The behavior of the system has been analyzed widi and without Zn(II). Fluorescence measurements indicate that binding with common monosaccharides (D-fructose, D-glucose and d-galactose) are not affected by incorporation of a Zn(II), whereas binding with uronic (D-glucuronic and D-galacturonic) adds and sialic (N-acetylneuraminic) acid are... 

Figure 9. Typical fluorescence signals obtained from a suspension of isolated rat cardiac myocytes after the application of maitotoxin (MTX). The arrow indicates the addition of MTX (10 g/mL), a detergent Emulgen 810 (1%), which frees all vesicular Ca , or EGTA (3.5 mM), a chelator that removes all free Ca in the cuvette. The intensity of Quin 2 fluorescence is expressed in arbitrary units. (Reproduced with permission from Ref. 20. Copyright 1987 Elsevier)...

The interest in colour indicators has recently increased as they are used for the direct determination of pH (acid-base indicators) and free calcium ions (fluorescent derivatives based on the calcium chelator EGTA as metallochromic indicators) in biological systems at cellular level. 

A calcium ion indicator dye (based on the structure of the chelator EGTA) that exhibits a strong fluorescence at 385 nm and can be used to measure changes in intracellular Ca concentration. The approximate dissociation constant for the Ca -Fura-2 complex is 0.1 juM, depending on cellular ion composition and pH. An esteri-fied derivative of Fura-2 readily crosses the peripheral membrane of many cells and, after hydrolysis, the release of Fura-2 permits calcium ion measurements within cells. See Calcium Ion Indicator Dyes Metal Ion Complex-ation... 

Metlay (145) studied the fluorescent decay of both EuD3 and EuD4. These compounds were prepared by the method of Crosby et al. (142). As Metlay points out, Whan and Crosby (146) in a later paper describe their preparation in more detail. They indicate that a period of heating in vacuum is necessary to convert the chelate from one containing four molecules of dibenzoylmethane per atom of europium to one containing but three. 

Fig. 1. Various schematics of bead display for molecular assemblies on beads. The Py subunits of the G protein (circles labeled with [i and y) are fused with either FLAG or hexahistidine tag, which recognizes the biotinylated M2 anti-FLAG antibodies on streptavidin-coated beads or chelated nickel on the dextran-treated beads. A socket and plug connecter is utilized to depict the very high-affinity interaction of the epitope tag. This modular setup allows for either a subunit (for capturing FPR-GFP) or as subunit (for capturing / 2AR-GFP) to be coupled with the fly subunit to form the complete G protein coating the bead. Fluorescent components such as GFP or ligand are indicated in green. See text for details.

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

Ca2+ levels, with one of the intracellular calcium-chelating, fluorescent probes, quin-2, fura-2 or indo-1, demonstrates that, in both cases, there is a rapid rise in intracellular Ca2+ as this ion is released from intracellular stores. Analysis of stimulated B lymphocytes, using the probe fura-2, indicates that if Ca2+ in the external medium is removed the intracellular Ca2+ level returns to basal levels in 5 to 7 minutes, but if there is Ca2+ present in the external media a sustained increase in intracellular Ca2+ is detected [36]. Such analysis suggests the opening of a plasma membrane calcium channel but the nature of the channel or mechanism of its opening are not presently known. It is possible that the opening of this channel could be stimulated by one of the inositol phosphates. 

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