Calcium complexes, fluorescence

Figure 2. Reporting of cytosolic free calcium levels by indo-1. Increases in cytosolic calcium, due either to entry of extracellular calcium via calcium channels or to release of intracellular calcium sequestered in organelles such as smooth endoplasmic reticulum, results in formation of the indo-l-calcium complex. Fluorescence intensity at 400 nm (excitation at 340 nm) is proportional to the concentration of this complex the dissociation constant for this complex is about 250 nff (24), making this probe useful for detecting calcium activities in the range of 25 to 2500 nJ. ...

The Calcein-calcium complex fluoresces in ultraviolet radiation, but the uncomplexed Calcein does not. The sample to be titrated is placed in a dark room or in a box and exposed to uv radiation. The sample then is titrated until the fluorescence disappears. This is a very sharp endpoint and must be approached slowly near the end. The color of the fluorescence is enhanced by viewing though an amber glass or a piece of yellow cellophane, which removes the small amounts of visible blue light from the lamp. 

There have been several papers concerned with the analytical uses of fluorescence. Thus the determination of lead in blood by atomic fluorescence methods,359 the determination of mercury at picogram l-1 levels in water,380 the use of isocein as a fluorescent reagent for calcium,361 fluorescent reactions of eriochrome red with Be, Mg, Al, In, Ga, and Zn as a method of determination,362 fluorimetric determination of chloroquine363 and of anthracene in anthraquinone,364 and the use of fluorescence methods in the characterization of complex mixtures 365 have been discussed. Readers with criminal tendencies... 

Figure 3.29 shows file fluorescence emission spectra of lO pM Quin-2 at different concentrations of calcium (0 to 40 pM) in the absence (A) and in the presence (B) ol 15.3 pM human serum albumin. From the data of figure 3.29, the authors generate steady-state emisaon spectra of the protein (Fig. 3.30a), Quin-2 (Fig. 3.30b), file Quin-2-calcium complex (Fig. 3.30c) and the Quin-2 - protein complex (Fig. 3.30d). One can notice that Quin-2 fluorescence emission spectrum in presence ofhuman serum albumin is blue slufted compared to Quin-2 flee in solution. The Quin-2-calcium complex is however red-shifted.

Fluorimetric methods have proven useful for the assay of metal ions in solution (i) for example, in vivo studies of calcium-selective fluorescence probes have been reported by Tsien (2). Most such analytical methods reported to date involve complexation of metal ions with aromatic heterocyclic ligands ("intrinsic" fluoroionophores). In 1977, Sousa described the synthesis of naphthalene-crown ether probes (Figure 1) in which the fluorophore 7C-system is insulated from the... 

We studied the calcium—EDTA chelation using Calcein [4] as the fluorescent indicator. This indicator forms a fluorescent complex with free calcium ions. In order to determine calcium in a sample, the excess EDTA remaining after stoichiometric chelation of calcium in the sample is "back-titrated" with a known concentration of calcium solution. The equivalence point is reached when all of the excess EDTA is chelated and is detected by fluorescence of the Calcein-calcium complex formed by a slight excess of free calcium ions. The calcium content of the sample (in micrograms) is equal to the volume (in milliliters) of titrant added when no sample was introduced, minus the volume (in milliliters) of titrant added when the sample was introduced, times the calcium content of the titrant (in micrograms per milliliter). 

The proportion of hydrochloric acid in the mobile phase was not to exceed 20%, so that complex formation did not occur and zone structure was not adversely affected. An excess of accompanying alkaline earth metal ions did not interfere with the separation but alkali metal cations did. The hthium cation fluoresced blue and lay at the same height as the magnesium cation, ammonium ions interfered with the calcium zone. 

Figure 3. Reporting of intracellular calcium sequestration by chlorotetracycline (CTC). CTC preferentially partitions into cell membranes and its fluorescence in this environment is sensitive to calcium bound to the membrane therefore its signal (excitation AOO nm, emission 530 nm) will come largely from organelles that bind or sequester calcium, such as smooth endoplasmic reticulum or mitochondria. Release of calcium from such organelles is accompanied by dissociation of the calcium-CTC complex, a decrease in CTC fluorescence and efflux of unbound probe from the organelle and from the cell.

This dye fluoresces after binding Pb+2 and Ca+2 lead is considered an interferant to the determination of calcium by this approach. However, by complexing the divalent lead ion with the heavy metal chelator TPEN (N,N,N ,N -tetrakis(2-pyridylmethyl)ethylene-diamine) prior to the addition of the fluo-3, the fluorescent... 

The natural fluorescence of CTC and its derivatives has been used extensively to determine small amounts of CTC in biological materials. Kohn (86) showed that the fluorescent complex formed by CTC with calcium ions and barbital could be extracted from animal tissues into an organic solvent and then measured spectrofluorometrically. The intense fluorescence of anhydro-CTC was used by Hayes and DuBuy (87) to determine CTC in animal tissues, tissue culture cells, and bacteria. Poiger and Schlatter (88) extracted CTC from biological material into ethyl acetate as the CTC-calcium trichloroacetate ion pair. The fluorescence of the antibiotic was then enhanced by the addition of magnesium ions and a base. 

The tyrosinate fluorescence observed with bovine testes calmodulin is argued to be due to tyrosinate in the ground state.(123) Of the two tyrosine residues in this calmodulin, Tyr-99 apparently has a low pKa near 7 for the formation of tyrosinate, which is most likely due to nearby side chains that are involved in calcium binding. These groups could then also account for the complex pH dependence of the 345-nm emission intensity. Besides the tyrosine and tyrosinate emissions at 305 and 345 nm, respectively, Pundak and Roche(123) also reported the existence of a third emission band between 312 and 320 nm. This band was similar in its pH and calcium dependence to the other residue, Tyr-138, and was speculated to be a result of a combination of contributions from the tyrosine and tyrosinate emissions. Since this band has its excitation profile shifted to the red, however, it could be that a hydrogen-bonded tyrosine exists in this calmodulin. Alternatively, it has also been found that the presence of the 345-nm emission depends upon the method of preparation (G. Sanyal, personal communication). 

C. Pigault, A. Follenius-Wund, B. Lux, and D. Gerard, A fluorescence spectroscopy study of the calpactin I complex and its subunits pit and p36 Calcium-dependent conformational changes, Biochim. Biophys. Acta 1037, 106-114 (1990). 

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... 

By introducing the complexing moiety —CH2N(CH2C02H)2 into fluorescein or umbelliferone metallochromic indicators termed calcein or fluorexone, and calcein blue or umbellicomplexone are formed. These indicators exhibit green (and blue) fluorescence in dilute alkali which disappears at pH 12 but reappears on the addition of calcium, strontium, or barium. Problems arise from the difficulty of freeing the indicators from fluorescent impurities, from self-fluorescence, and from the quenching effect of copper ions. 

Figure 1. (Bottom) Diagram of the electrostatic potential adjacent to a membrane bearing a positive charge. The zeta potential is the potential at the hydrodynamic plane of shear, which should be about 2 A from the surface of the membrane. (Top) Schematic of the location of the probe molecules used to detect the potential produced by the adsorption of calcium and other alkaline earth cations to membranes formed from PC. The divalent cation cobalt and the amphipathic, anionic, fluorescent probe TNS will sense the potential at the interface. The non-actin-Rf complex will sense the potential in the center of the membrane.

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