Luminescence intensity

Electron-transfer reactions producing triplet excited states can be diagnosed by a substantial increase in luminescence intensity produced by a magnetic field (170). The intensity increases because the magnetic field reduces quenching of the triplet by radical ions (157). 

Application of a modified sorbent is preferable, since in this case the intensity luminescence (/) of Ln, as well as the rate of its determination is higher about 6-7 times. The comparison of luminescence intensity of Ln -ligand complex solution before the soi ption with results of I after soi ption by both non-modified and modified PMMA showed that I increased in 30 and about 200 times, respectively. 

On the fig. 1 is given the family of curves displaying the dependence of chemi-luminescence intensity ver-sus time at injection of AO (the arrow shows the moment of inhibitor injection). 

From figure follows, that in case of injection of small concentration of AO in it will be consume rather slowly, that results in slow variation of luminescence intensity during certain time. 

It has been established, that both DN and Ibp form complex compounds with ions Eu(III), Sm(III), Tb(III) and Dy(III), possessing luminescent properties. The most intensive luminescence is observed for complex compounds with ion Tb(III). It has been shown, that complexation has place in low acidic and neutral water solutions at pH 6,4-7,0. From the data of luminescence intensity for the complex the ratio of component Tb Fig was established equal to 1 2 by the continuous variations method. Presence at a solution of organic bases 2,2 -bipyridil, (Bipy) and 1,10-phenanthroline (Phen) causes the analytical signal amplification up to 250 (75) times as a result of the Bipy (Phen) inclusion in inner coordination sphere and formation of different ligands complexes with component ratio Tb Fig Bipy (Phen) = 1 2 1. 

We detenuined the influence of oxy- and ketocarboxylic acids (succinate, fumarate, adipinate, a-ketoglutarate, isocitrate, tartrate, E-malate) on the luminescence intensity of the Eu-OxTc complex. These substances interact as polydentate ligands similarly to citrate with the formation of ternary complexes with Eu-OxTc. As to succinate, fumarate, adipinate and a-ketoglutarate this they cannot effectively coordinate with EiT+ and significant fluorescence enhancement was not observed. 

It was found that the effect of solvents and various surfactants Triton X-100, Twin-80, Brij-35 sodium laurylsulfate, sodium cetylsulfate, cetylpyridinium chloride, cetyltrimethylammonium bromide on the luminescence intensity is insignificant. 

The optimal conditions for the complexation were found. The luminescence of Tb " in (L ) complex was established to observed in a range of pH 2,0-11,0 with maximum at 7,0-7,5. The Tb (III) luminescence in complex with (L ) aslo depends on amount of reagents, solvent nature, amount of surfactants and trioctylphosphinoxide (TOPO). It was shown that introduction into the system Tb-L the 3-fold excess sodium dodecylsulfate (SDS) increases the luminescence intensity by 40 times and introduction into the system Tb-L the 3-fold excess TOPO increases the luminescence intensity by 25 times by the order value connecting with the crowding out of water molecules from the inner sphere of complexes. 

Figure 15-15. Relative luminescence intensity (open markers) compared with pristine MEH-PPV of composite films of MEH-PPV with CM and a series of TCNQ-like acceptors 1-6 (see Fig. 15-2 lor abbreviations) as a function of their reduction potentials at 80 K. Riglil-liand axis shows the ratio of the photoinduced absorption intensity of the bands at 1.34 and 1.22 eV (solid markers) (reproduced by permission of the American Institute of Physics from Ref. 1871).

The optimum pH for the luminescence reaction is about 7.8, and the luminescence intensity is strongly affected by the buffer salt used... 

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.

Relationship between Ca2+ concentration and luminescence intensity. In the measurement of Ca2+ concentration with aequorin, the calibration of the relationship between Ca2+ concentration and luminescence intensity is essential. However, the application of this relationship is complicated by the chelator used to set the Ca2+ concentration, for the reason noted above. To minimize the complication, we used only a minimum amount of EDTA to protect aequorin in the measurements to obtain the relationship between Ca2+ -concentration and light intensity, and plotted the data as shown in Fig. 4.1.7 (Shimomura and Johnson, 1976). The concentration of EDTA was... 

Fig. 4.1.8 Influence of various calcium chelators on the relationship between Ca2 " concentration and the luminescence intensity of aequorin, at 23-25°C (panel A) in low-ionic strength buffers (I < 0.005) and (panel B) with 150 mM KC1 added. Buffer solutions (3 ml) of various Ca2+ concentrations, pH 7.05, made with or without a calcium buffer was added to 2 pi of 10 pM aequorin solution containing 10 pM EDTA. The calcium buffer was composed of the free form of a chelator (1 or 2mM) and various concentrations of the Ca2+-chelator (1 1) complex to set the Ca2+ concentrations (the concentration of free chelator was constant at all Ca2+ concentrations). The curves shown are obtained with 1 mM MOPS (A), 1 mM gly-cylglycine ( + ), 1 mM citrate (o), 1 mM EDTA plus 2mM MOPS ( ), 1 mM EGTA plus 2 mM MOPS ( ), 2 mM NTA plus 2 mM MOPS (V), and 2 mM ADA plus 2 mM MOPS (A). In the chelator-free buffers, MOPS and glycylglycine, Ca2+ concentrations were set by the concentration of calcium acetate. Reproduced with permission, from Shimomura and Shimomura, 1984. the Biochemical Society.

The second procedure is to measure the luminescence intensities at various Ca2+ concentrations and plot log (light intensity) against —log [Ca2+] for each aequorin. Examples of this method are shown in Fig. 4.1.14. This method provides more detailed information on the sensitivity of each aequorin. Generally, an increase in Ca2+ sensitivity shifts the curve to the left. 

Properties of the luciferases. According to Shimomura and Flood (1998) and Shimomura et al. (2001), all Periphylla luciferases L, A, B and C catalyze the oxidation of coelenterazine, resulting in the emission of blue light (Amax 465 nm). Luciferases B (40 kDa) and C (80 kDa) are apparently the dimer and tetramer, respectively, of luciferase A (20 kDa). The presence of a salt is essential for the activity of luciferase, and the optimum salt concentration is about 1M in the case of NaCl for all forms of luciferases. The luminescence intensity of luciferase L is maximum near 0°C, and decreases almost linearly with rising temperature, falling to zero intensity at 60°C the luminescence intensity profiles of luciferases A, B and C show their peaks at about 30°C (Fig. 4.5.3). The Michaelis constants estimated for luciferases A, B and C with coelenterazine are all about 0.2 xM, and that for luciferase L is 1.2 jiM. 

The spectra of the luminescence of coelenterazine catalyzed by recombinant Renilla luciferase in the presence and absence of Renilla GFP are shown in Fig. 4.6.3 (Lorenz et al., 1991). Note that the luminescence intensity at the emission peak is increased more than... 

Luciferase activity on e-coelenterazine. In the presence of Renilla luciferase, the luminescence intensity of e-coelenterazine is more than 5 times higher than that of coelenterazine under the same conditions... 

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. 

Assay of photoprotein. The activity of the photoprotein was measured in 1ml of 20 mM Tris-HCl buffer, pH 8.0, containing 0.6 M NaCl at room temperature. The intensity and total amount of light emitted were recorded. The luminescence intensity is markedly intensified by adding 5 il of catalase solution (crystalline bovine liver catalase 1.5 mg/ml) and 10 pi of 3% H2O2. 

Fig. 7.2.8 Influence of pH on the luminescence intensity of the Odontosyllis luciferin-luciferase reaction at room temperature. From Shimomura et ai, 1963d, with permission from John Wiley Sc Sons Ltd.

Luciferase-catalyzed luminescence of luciferin. Odontosyllis luciferin emits light in the presence of Mg2+, molecular oxygen and luciferase. The relationship between the luminescence intensity and the pH of the medium shows a broad optimum (Fig. 7.2.8). The luminescence reaction requires a divalent alkaline earth ion, of which Mg2+ is most effective (optimum concentration 30 mM). Monovalent cations such as Na+, K+, and NH have little effect, and many heavy metal ions, such as Hg2+, Cu2+, Co2+ and Zn2+, are generally inhibitory. The activity of crude preparations of luciferase progressively decreases by repeated dialysis and also by concentrating the solutions under reduced pressure. However, the decreased luciferase activity can be completely restored to the original activity by the addition of 1 mM HCN (added as KCN). The relationship between the concentration of HCN and the luciferase activity is shown in Fig. 7.2.9. Low concentrations of h and K3Fe(CN)6 also enhance luminescence, but their effects are only transient. 

Fig. 9.2 Changes in the luminescence intensity, the contents of luciferin and SOD, and medium pH during the mycelial growth of five kinds of bioluminescent fungi Panellus stipticus, Armillaria mellea, Mycena citricolor, Pleurotus japonicus, and Omphelotus olearius. The ordinate readings of the curves marked 1/3 should be multiplied by 3. For the SOD activity, see Table 9.2, footnote e. From Shimomura, 1992, with permission from Oxford University Press.

Difference between fruiting body and mycelium. The luminescence intensities of both fruiting bodies and mycelia are in a relatively narrow range (6-21 x 1010 quanta s 1 g1), despite the clear difference that the fruiting bodies are generally rich in luciferin and the mycelia are rich in SOD (Table 9.2). 

Assay methods. Activity can be measured at pH 5.5 or at pH 8.0. With the same sample, the pH 5.5 method gives a much higher luminescence intensity than the pH 8.0 method (and, shorter reaction time), although the total amounts of light emitted by the two methods are practically equal (Fig. 9.4). The pH 5.5 method is susceptible to inhibition by various salts, whereas the pH 8.0 method is not affected. 

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