Tris luminescence

Examples include luminescence from anthracene crystals subjected to alternating electric current (159), luminescence from electron recombination with the carbazole free radical produced by photolysis of potassium carba2ole in a fro2en glass matrix (160), reactions of free radicals with solvated electrons (155), and reduction of mtheiiium(III)tris(bipyridyl) with the hydrated electron (161). Other examples include the oxidation of aromatic radical anions with such oxidants as chlorine or ben2oyl peroxide (162,163), and the reduction of 9,10-dichloro-9,10-diphenyl-9,10-dihydroanthracene with the 9,10-diphenylanthracene radical anion (162,164). Many other examples of electron-transfer chemiluminescence have been reported (156,165). 

Luminescence reaction (Viviani et al., 2002a) The luciferin-luciferase luminescence reaction was carried out in 0.1 M Tris-HCl, pH 8.0, containing 2mM ATP and 4mM Mg2+. Mixing luciferase with luciferin and ATP resulted in an emission of light with rapid onset and a kinetically complex decay. Further additions of fresh luciferase, after the luminescence has decayed to about 10% of its maximum value, resulted in additional luminescence responses similar to the initial one (Fig. 1.15). According to the authors, the repetitive light emission occurred in consequence of the inhibition of luciferase by a reaction product, as seen in the case of the firefly system (McElroy et al., 1953). The luminescence spectrum showed a peak at 487nm (Fig. 1.16). 

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. 3.3.1 Luminescence spectrum of coelenterazine catalyzed by the luciferase of the decapod Oplophorus in 15 mM Tris-HCl, pH 8.3, containing 50 mM NaCl (solid line). For comparison, the luminescence catalyzed by the luciferase of the anthozoan sea pansy Renilla is shown with dashed line (in 25 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl).

Quantum yield and luciferase activity The quantum yield of coelenterazine in the luminescence reaction catalyzed by Oplophorus luciferase was 0.34 when measured in 15 mM Tris-HCl buffer, pH 8.3, containing 0.05 M NaCl at 22°C (Shimomura et al., 1978). The specific activity of pure luciferase in the presence of a large excess of coelenterazine (0.9pg/ml) in the same buffer at 23°C was 1.75 x 1015 photons s 1 mg-1 (Shimomura et al., 1978). Based on these data and the molecular weight of luciferase (106,000), the turnover number of luciferase is calculated at 55/min. 

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.

Freeze-drying of aequorin. The process of freeze-drying always results in some loss in the luminescence activity of aequorin. Therefore, aequorin should not be dried if a fully active aequorin is required. The loss is usually 10% or more. The loss can be somewhat lessened by adjusting the buffer composition the use of 100 mM KCl and some sugar (50-100 mM) seems to be beneficial. The buffer composition used at the author s laboratory is as follows 100 mM KCl, 50 mM glucose, 3 mM HEPES, 3 mM Bis-Tris, and at least 0.05 mM EDTA, pH 7.0. 

Fig. 4.1.4 Influence of pH on the total light emission and initial light intensity of aequorin. Buffer solutions containing 0.1 mM calcium acetate, 0.1 M NaCl, and 10 mM sodium acetate (for pH < 7) or 10 mM Tris-HCl (for pH > 7) were adjusted to various pH with acetic acid or NaOH, and then 2 ml of the solution was added to 3 pi of aequorin solution containing 1 mM EDTA to elicit luminescence, at 22°C. The data shown are a revision of Fig. 9 in Shimomura et al., 1962. The half-total time is the time required to emit 50% of total light.

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.

Fig. 4.3.1 Effect of pH on the total light emission of phialidin (A), and the temperature stability profiles of phialidin (minute open circles) and aequorin (solid line) (B). In A, each buffer contained 0.1 M CaCl2 plus 0.1 M Tris, glycine or sodium acetate, the pH being adjusted with NaOH or HC1. In B, the photoprotein samples in 10 mM Tris-EDTA buffer solution, pH 8.0, were maintained at a test temperature for 10 min, and immediately cooled in an ice water bath. Then total luminescence activity was measured by injecting 1ml of 0.1 M CaCl2/Tris-HCl, pH 7.0, to 10 pd of the test solution. From Levine and Ward (1982), with permission from Elsevier.

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. 

Fig. 4.5.3 Effect of temperature on the light intensity of coelenterazine catalyzed by Periphylla luciferases A, B, C and L, in 3 ml of 20 mM Tris-HCl, pH 7.8, containing 1 M NaCl and 0.05% BSA. The luminescence reaction was started by the addition of 10 (xl of 0.1 mM methanolic coelenterazine. The amounts of luciferase used for the measurement of each point luciferase A, 170 LU luciferase B, 190 LU luciferase C, 210 LU luciferase L, 210 LU. From Shimomura et al., 2001.

Fig. 4.5.5 Effect of pH on the luminescence of coelenterazine catalyzed by Periphylla luciferases A, B and C, and on the stability of the luciferases. The effect on light intensity (solid lines) was measured in 3 ml of 50 mM phosphate buffers, pH 4.1-7.25, and 50 mM Tris-HCl buffers, pH 7.1-9.7, all containing 1 M NaCl, 0.025% BSA, and 0.3 pM coelenterazine. To measure the stability (dotted lines), a luciferase sample (5 pi) was left standing for 30 min at room temperature in 0.1 ml of a buffer solution containing 1 M NaCl and 0.025% BSA and having a pH to be tested, and then luciferase activity in 10 pi of the solution was measured in 3 ml of 20 mM Tris-HCl, pH 7.8, containing 1M NaCl, 0.05% BSA, and 0.3 pM coelenterazine at 24°C. The amounts of luciferases used for measuring each point were luciferase A, 150 LU luciferases B and C, 170 LU. One LU = 5.5 x 108 quanta/s. From Shimomura etal., 2001.

Fig. 5.8 Influence of pH, temperature, NaCl concentration, and the concentration of coelenterazine on the light intensity of luminescence reaction catalyzed by the luciferases of Heterocarpus sibogae, Heterocarpus ensifer, Oplophorus gracilirostris, and Ptilosarcus gruneyi. Buffer solutions used 20 mM MOPS, pH 7.0, for Ptilosarcus luciferase and 20 mM Tris-HCl, pH 8.5, for all other luciferases, all with 0.2 M NaCl, 0.05% BSA, and 0.3 p,M coelenterazine, at 23°C, with appropriate modifications in each panel. Various pH values are set by acetate, MES, HEPES, TAPS, CHES, and CAPS buffers.

Fig. 6.2.4 Change in the absorption spectrum of pholasin (14.5 p,M) caused by the luminescence reaction catalyzed by Pholas luciferase (1.1 p.M). The curve shown is the differential spectrum between a cell containing the mixture of pholasin and Pholas luciferase (0.9 ml in the sample light path) and two cells containing separate solutions of pholasin and the luciferase at the same concentrations (in the reference light path), all in 0.1 M Tris-HCl buffer, pH 8.5, containing 0.5 M NaCl. Four additions of ascorbate (3 iM) were made to the sample mixture to accelerate the reaction. The spectrum was recorded after 120 min with a correction for the base line. From Henry and Monny, 1977, 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.

Fig. 6.3.7 Luminescence spectrum of a homogenate of the luminous organ of Symplectoteuthis oualaniensis in the presence of 0.5 M KC1 (from Tsuji and Leisman, 1981). A homogenate suspension (1 ml) and 1MKC1 (1 ml), both made with 50 mM Tris-HCl, pH 7.6, containing 1 mM dithioerythritol, were mixed and the spectrum was measured 6 min after mixing. Note that the luminescence of the photoprotein symplectin isolated from the luminous organs showed a maximum at 470—480 nm (Takahashi and Isobe, 1993, 1994).

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. 7.1.3 Influence of the buffer and the type of peroxide on the luminescence reaction of Chaetopterus photoprotein. The reaction was initiated at zero time by the addition of a peroxide (old dioxane or H2O2) and FeSC>4 in each case, with successive additions of FeSC>4 or H2O2 at the time indicated with an arrow. In the experiments of the two upper curves, 10 pi of old dioxane and 1 pi of lOmM FeSC>4 were added at zero time, followed by 1 pi of 10 mM FeSC>4 at each arrow. In the experiments of the two lower curves, 50 pi of 10 mM H2O2 and 20 pi of 10 mM FeSC>4 were added at zero time, followed by 50 pi of 10 mM H2O2 or 20 pi of 10 mM FeSC>4 at each arrow. All in 5 ml of 10 mM phosphate or Tris buffer, pH 7.2. From Shimomura and Johnson, 1966.

Fig. 7.1.5 Fluorescence spectra of purified Chaetopterus photoprotein (CPA) in 10 mM ammonium acetate, pH 6.7 (solid lines), and the bioluminescence spectrum of the luminous slime of Chaetopterus in 10 mM Tris-HCl, pH 7.2 (dashed line). Note that the luminescence spectrum of Chaetopterus photoprotein in 2 ml of 10 mM Tris-HCl, pH 7.2, containing 0.5 M NaCl, 5 pi of old dioxane and 2 pi of 10 mM FeSC>4 (Amax 453-455 nm) matched exactly with the fluorescence emission spectrum of the photoprotein. No significant change was observed in the fluorescence spectrum after the luminescence reaction.

Purified LBP is obtained from the crude LBP separated in the gel filtration of the 35 kDa luciferase on Sephadex G-100 (see Fig. 8.2). The fractions of crude LBP are combined and the protein is precipitated with ammonium sulfate (75% saturation). The precipitate is dissolved in a small volume of lOmM Tris-HCl/5 mM 2-mercaptoethanol, pH 8, and a small amount of luciferin is added as a tracer. Then, the crude LBP is purified on a column of Sephadex G-200 (Hastings and Dunlap, 1986). The fractions of LBP are identified by luminescence produced by the addition of luciferase at pH 6.3 the luminescence due to the tracer luciferin is proportional to the amount of LBP in each fraction. 

Assay of luminescence activity. A methanolic solution of the activation product (5-50 xl) is mixed with 3 ml of 50 mM Tris-HCl buffer, pH 7.8, containing 0.18 mM EDTA and about 5 mg of CTAB. After leaving the solution for a few minutes, 15 (rl of 50 mM FeS04 and 30 il of-10% H2O2 are added in this order. The light emission begins when H2O2 is added. 

Fig. 9.9 Luminescence spectrum of a young fruiting body of Fanellus stipticus (1) the chemiluminescence spectra of PM-1 in the presence of CTAB (2) hexadecanoyl-choline iodide (3) and tetradecanoylcholine chloride (4). Chemiluminescence was elicited with Fe2+ and H2O2 in 50mM Tris buffer, pH 8.0, containing 0.18mM EDTA.

Assay of Ophiopsila photoprotein. The luminescence reaction is initiated by the injection of 0.1 ml of 1% H2O2 into 3 ml of Tris-HCl buffer, pH 7.5, containing 0.5 M NaCl and 10-100 pi of a sample solution, and the total light emission is measured. When assaying the... 

Assay of Luminodesmus photoprotein. A sample solution (10 100pl) is mixed with 2ml of 10 mM Tris buffer, pH 8.5, containing 0.1 mM ATP and 1 mM MgCl2, and the peak intensity of the luminescence is measured. After mixing, the light intensity reaches its maximum in a few seconds, then gradually diminishes in... 

Fig. 10.2.2 Influence of the concentrations of ATP, Mg2-1- and Ca2+ on the maximum luminescence intensity of the photoprotein of the millipede Luminodestnus. The luminescence reaction was started by mixing a solution of the photoprotein (A280 0.3, 10 pi) with 2 ml of 10mM Tris-HCl buffer, pH 8.3, containing either 1 mM MgCb plus various concentrations of ATP or 0.05 mM ATP plus various concentrations Mg2+ or Ca2+. From Shimomura, 1981, with permission from the Federation of the European Biochemical Societies.

Assay of luminescence activity. Luciferin solution (1 ml) is mixed with 1.2 ml of 0.5 M Tris buffer (pH 8.2) and 0.3 ml of luciferase solution. The luminescence reaction is initiated by the injection of 0.5 ml of 0.176 mM H2O2 to the luciferin-luciferase mixture. The light emission is characterized by a flash of light, followed by a rapid decay to a much lower steady-state level (Fig. 10.4.1). The maximum light intensity of the flash is taken as the measure of the luminescence activity. 

Fig. 10.4.2 The effects of temperature (left panel) and pH (right panel) on the peak intensities of the Balanoglossus luminescence reaction. In the measurements of the temperature effect, 0.5 ml of 0.176 mM H2O2 was injected into a mixture of 1.2 ml of 0.5 M Tris buffer (pH 8.2), 0.3 ml of luciferase, and 1 ml of luciferin, at various temperatures. For the pH effect, the Tris buffer (pH 8.2) was replaced with the Tris buffers and phosphate buffers that have various pH values, and the measurements were made at room temperature. From Dure and Cormier, 1963, with permission from the American Society for Biochemistry and Molecular Biology.

The amount of raw material required for the chemical study of luminescent substances today is much less than that required 20 or 30 years ago, due to the advance in instrumentation. In the case of a luciferin, try to obtain 1-2 mg of purified luciferin. If that is not feasible, try to obtain at least 0.1 mg of purified material, and with some luck, the structure may be obtained. 

A sterically hindered homoleptic samarium(lll) tris(amidinate), Sm[HC (NC6H3Pr2-2,6)2]3, was obtained by oxidation of the corresponding Sm(II) precursor (cf. Scheme 54). Magnetic data and the results of low temperature absorption, luminescence, and magnetic circular dichrosim spectra have been... 

Self-assembly of aromatic dendron subunits has been tried by the design of coordination to multivalent metal cations (i.e., metal-cored dendrimer complexes). Several metal-cored dendrimer complexes have successfully exhibited luminescence by antenna effects. 

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