Luminescence.measurements, with

Fig. 2.4 The spectrum of bacterial luminescence measured with B. harveyi luciferase, FMN, tetradecanal and NADH, in 50 mM phosphate buffer, pH 7.0, at 0°C (dashed line from Matheson et al., 1981) and the absorption and fluorescence emission spectra of LumP (solid lines) and Rf-LumP (dotted lines) obtained from P. leiog-natbi, in 25 mM phosphate buffer, pH 7.0, containing 1 mM EDTA and 10 mM 2-mercaptoethanol, at room temperature (from Petushkov et al, 2000, with permission from Elsevier). LumP is a lumazine protein, and Rf-LumP contains riboflavin instead of lumazine in the lumazine protein. Fluorescence emission curves are at the right side of the absorption curves.

Fig. 4.1.5 The time course of aequorin luminescence measured with various concentrations of Ca2+. Calcium acetate solution (5 ml) was added to 10 pi of aequorin solution to give the final Ca2+ concentrations of 10 2 M (A), 10-4 M (B), 10-5 M (C), 10 6 M (D), and 10 7 M (E) at 25°C. The dashed line (F) represents the light emitted following the addition of deionized distilled water that had been redistilled in quartz. The concentration of EDTA derived from the aequorin sample was 10 7 M (final cone.). From Shimomura et al., 1963b, with permission from John Wiley Sons Ltd.

Luminescence Measurements with an Intensified Diode Array... 

The use of fluorescent organometallic complexes to label biological substrates is beginning to provide some exciting alternatives to the more traditional organic dyes [126], with suitable iridium [127-129], rhodium [130], platinum [131], rhenium [132-135] and osmium [136] examples having recently been reported. The Re diimine wires (13g and 13h, Fig. 13), have been shown to form complexes with the nitric oxide synthase mutant 5114 [137]. Steady-state luminescence measurements with 13h establish a dissociation constant of 100 nM, while 13h binds with a... 

Chemiluminescent analyzers are based on the light (chemiluminescence) emitted in the gas-phase reaction of ozone with ethylene, which is measured with a photomultipHer tube. The resulting current is proportional to the ozone concentration (see Luminescent materials, chemiluminescence). 

The bioluminescence systems of Phengodidae (railroad worms) and Elateroidae (click beetles) are basically identical to that of Lampyridae (fireflies), requiring firefly luciferin, ATP, Mg2+ and a luciferase for light emission. However, there seem to be some differences. Viviani and Bechara (1995) reported that the spectra of the luminescence reactions measured with the luciferases of Brazilian fireflies (6 species) shift from the yellow-green range to the red range with lowering of the pH of the medium, like in the case of the Photinus pyralis luciferase (see Section 1.1.5), whereas the spectra... 

Energy transfer to fluorescent proteins. There are marked differences among the various bacterial species and strains in terms of the in vivo luminescence spectra. The emission maxima are spread mostly in a range from 472 to 505 nm (Seliger and Morton, 1968), but one of the strains, P. fischeri Y-l, shows a maximum at 545 nm (Ruby and Nealson, 1977), as shown in Fig. 2.3. However, the in vitro luminescence spectra measured with purified luciferases obtained from the various bacterial species and strains are all similar (Amax about 490 nm). The variation in the in vivo luminescence spectra may be due to the occurrence of an intermolecular energy transfer that increases the efficiency of light emission. 

Fig. 3.2.7 Left panel Effects of temperature on the luminescence intensity and stability of the protein P from Meganyctiphanes. The initial light intensity was measured with F plus P in 5 ml of 20 mM Tris-HCl/0.15 M NaCl, pH 7.5, at various temperatures. In the stability test, P was kept at the indicated temperature for 10 min, then mixed with 5 ml of 25 mM Tris-HCl/1 M NaCl, pH 7.59, containing F, to measure initial light intensity. Right panel Effect of the concentration of salts on the light intensity of the luminescence of F plus P, in 25 mM Tris-HCl, pH 7.6, at near 0°C. In the case of NaCl, the light intensity decreased to about a half after 10 min. From Shi-momura and Johnson, 1967, with permission from the American Chemical Society.

Fig. 3.3.3 Effects of temperature on the activities of luciferase ( ) and the quantum yields of coelenterazine (o) in the Oplophorus bioluminescence reaction. The activity was measured with coelenterazine (4.5 pg) and luciferase (0.05 pg), and the quantum yields with coelenterazine (0.2 pg) and luciferase (200 pg), in 5 ml of 15 mM Tris-HC1 buffer, pH 8.3 (at 25°C), containing 50 mM NaCl. Coelenterazine was first added to the buffer solution at the designated temperature, then the luminescence reaction was started by a rapid injection of 0.1 ml of luciferase solution. Replotted from Shimomura et al., 1978, with permission from the American Chemical Society.

Fig. 4.1.11 Influence of the concentration of apoaequorin on the yield of regenerated aequorin after 12 h at 4°C (solid line), and on the initial light intensity of the apoaequorin-catalyzed luminescence of coelenterazine (dashed line). The regenerated aequorin was measured with a 10 pi portion of a reaction mixture (0.5 ml) made with 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 5 mM 2-mercaptoethanol, 10 pi of methanolic 0.6 mM coelenterazine, and various amounts of apoaequorin. The luminescence activity of apoaequorin was measured in 2 ml of 10 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl, 2 mM CaCb, 2 mM 2-mercaptoethanol, 10 pi of methanolic 0.2 mM coelenterazine, and various amounts of apoaequorin. Reproduced with permission, from Shimomura and Shimomura, 1981. the Biochemical Society.

Luminescence activity. The specific luminescence activities (quanta/s emitted from 1ml of a solution of A280nm,icm 1.0) of luciferases A, B and C are in a range of 1.2 4.1 x 1016 photons/s when measured with the standard assay buffer (20 mM Tris-HCl, pFl 7.8, containing 1M NaCl, 0.05% BSA, and 0.14 xg/ml of coelenterazine, at 24°C). These are the highest specific activities of coelenterazine luciferases. 

Inouye and Shimomura, 1997). With Ptilosarcus luciferase, the luminescence intensity of e-coelenterazine is also significantly higher than that of coelenterazine. With other coelenterazine luciferases, however, the luminescence intensity of e-coelenterazine is generally lower than that of coelenterazine for example, the luminescence intensities of e-coeienterazine measured with the luciferases of the decapod shrimps, the jellyfish Periphylla, and the copepod Pleuromamma, were 50%, 4%, and 0.8%, respectively, in comparison with that of coelenterazine. Thus, the luminescence of coelenterazine catalyzed by Pleuromamma luciferase is suppressed by the addition of e-coelenterazine. 

Fig. 6.1.5 Fluorescence spectra of the purple protein (1-4) and the luminescence spectrum measured with Latia luciferin, luciferase and the purple protein (5 Xmax 536 nm). Excitation spectra (1) and (2) were measured with emission at 630 nm and 565 nm, respectively. Emission spectra (3) and (4) were measured with excitation at 285 nm and 380 nm, respectively. From Shimomura and Johnson, 1968c, with permission from the American Chemical Society.

Fig. 6.3.4 Luminescence spectrum of the Watasenia bioluminescence reaction measured with a crude extract of light organs that contain particulate matters, in chilled 0.1 M Tris-HCl buffer, pH 8.26, containing 1.5 mM ATP. From Tsuji, 2002, with permission from Elsevier.

Table 6.3.1 Luminescence of a partially purified sample of Symplectoteutbis lurni-nosa photoprotein (50 kDa fraction) measured with 20 pi of the photoprotein sample in 1 ml of 20 mM Tris-HCl buffer, pH 8.0, containing 0.6 M NaCl.a All at 20°C.

Fig. 7.3.2 Comparison between the in vivo luminescence spectrum of a freshly exuded slime of Diplocardia longa and the in vitro luminescence spectrum measured with partially purified preparations of Diplocardia luciferin and luciferase. Reproduced from Bellisario et al., 1972, with permission from the American Chemical Society. Note that the in vitro emission maximum shifts to 490 nm when a sample of pure luciferin is used (Ohtsuka et al., 1976).

According to the Kuwabara-Wassink paper, the purified luciferin in aqueous neutral buffer solution showed an absorption maximum at 320 nm, and a fluorescence emission peak at 490 nm. The luminescence emission maximum measured with Airth s fungal luciferase system was 524 nm at pH 6.5, whereas the chemiluminescence emission maximum of the luciferin with H2O2 plus a droplet of strong NaOH plus ferrous sulfate was 542 nm. No information was reported on the chemical nature of the luciferin. 

Minute amounts of coelenterazine can also be measured utilizing apoaequorin or apoobelin (Campbell and Herring, 1990 Thompson et ah, 1995). In this method, a sample containing coelenterazine is treated with an excess amount of apophotoprotein (apoaequorin or apoobelin) to convert it to a Ca2+-sensitive photoprotein (aequorin or obelin). The photoprotein formed is assayed by luminescing it with Ca2+ to determine the amount of coelenterazine originally existed. With this method, the luminescence reaction is fast and usually complete in a few seconds, in contrast to the slower luminescence reactions with luciferases that sometimes require a few minutes to complete. However, the formation of photoprotein from apoaequorin is slow and not necessarily quantitative, and the overall accuracy of the photoprotein method does not compare favorably with that of the luciferase method that directly measures coelenterazine. The author recommends using a luciferase if the enzyme is available. 

If a trace activity is indicated by the luminescence intensity measurement, the following two methods can be used to determine whether the light emission is due to the luciferase or it is an artifact (1) Measure the luminescence intensity with a buffer that contains 1 mM EDTA (add luciferase to this buffer and wait 1 min before mixing with luciferin). If the luminescence was caused totally by luciferase, the light intensity will be decreased to about 20% by EDTA (see Section 3.1.7). (2) Inactivate luciferase by acidifying the sample to pH about 2.0, followed by neutralization with NaHCC>3. Inactivated luciferase should not show any luciferase activity. 

The immense growth in the luminescence literature during the period between these two reviews had little to do with developments in fundamental theory. It was mainly due to the availability of new instrumentation, such as the photomultiplier (around 1950), the laser (around 1960), transistor and microcircuit electronics (around 1970), and ready access to laboratory computers (around 1975). All aspects of luminescence theory now being used to interpret luminescence measurements have been known since the early 1900 s and nearly all of the types of measurements now being made had been initiated with cruder techniques by 1930. We discuss here many of the latest techniques in luminescence analysis with selected highlights from the historical development of luminescence and a look at several recent developments in luminescence applications that appear likely to be important to future research. 

Many current multidimensional methods are based on instruments that combine measurements of several luminescence variables and present a multiparameter data set. The challenge of analyzing such complex data has stimulated the application of special mathematical methods (80-85) that are made practical only with the aid of computers. It is to be expected that future analytical strategies will rely heavily on computerized pattern recognition methods (79, 86) applied to libraries of standardized multidimensional spectra, a development that will require that published luminescence spectra be routinely corrected for instrumental artifacts. Warner et al, (84) have discussed the multiparameter nature of luminescence measurements in detail and list fourteen different parameters that can be combined in various combinations for simultaneous measurement, thereby maximizing luminescence selectivity with multidimensional measurements. Table II is adapted from their paper with the inclusion of a few additional parameters. 

The data were collected using fluorescence measurements, which allow both identification and quantitation of the fluorophore in solvent extraction. Important experimental considerations such as solvent choice, temperature, and concentrations of the modifier and the analytes are discussed. The utility of this method as a means of simplifying complex PAH mixtures is also evaluated. In addition, the coupling of cyclodextrin-modified solvent extraction with luminescence measurements for qualitative evaluation of components in mixtures will be discussed briefly. 

Alkaline phosphatase-labeled probes are synthesized so that 18 bases are complementary to sequences on the arms of the bDNA. Three hybridization sites are located on each branch for a total binding capacity of 45 labeled probes per bDNA molecule. The alkaline phosphatase catalyzes the dephosphorylation of chemiluminescent substrate, dioxetane (Lumi-Phos Plus, Lumigen, Detroit, MI). The intensity of the light emission is measured with a plate luminometer as relative luminescent units. 

Ultraviolet absorption spectra were obtained from a Cary 118C Spectrophotometer. Luminescence measurements were obtained from a Perkin-Elmer Model MPF-3 Fluorescence Spectrophotometer equipped with Corrected Spectra, Phosphorescence and Front Surface Accessories. A Tektronix Model 510N Storage Oscilloscope was used for luminescence lifetime measurements. Fiber irradiation photolyses were carried out in a Rayonet Type RS Model RPR-208 Preparative Photochemical Reactor equipped with a MGR-100 Merry-go-Round assembly. 

Figure 7 Luminescent lanthanide complexes with representative luminescence lifetimes, and hydration states (derived from luminescence measurements) where appropriate.

Cells are lysed for Firefly and Renilla luciferase assays using the Dual-Luciferase Reporter Assay system (Promega), following the manufacturer s instructions. We use a multimode microplate reader with automatic injectors (FLUOROstar Optima from BMG Labtech, OfFenburg, Germany) for luminescence measurements. 

More fluorescence features than just the emission intensity can be used to develop luminescent optosensors with enhanced selectivity and longer operational lifetime. The wavelength dependence of the luminescence (emission spectmm) and of the luminophore absorption (excitation spectrum) is a source of specificity. For instance, the excitation-emission matrix has shown to be a powerful tool to analyze complex mixtures of fluorescent species and fiber-optic devices for in-situ measurements (e.g. 

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