Coelenterazine-luciferase Reaction

The bioluminescence reaction of Oplophorus is a typical luciferin-luciferase reaction that requires only three components luciferin (coelenterazine), luciferase and molecular oxygen. The luminescence spectrum shows a peak at about 454nm (Fig. 3.3.1). The luminescence is significantly affected by pH, salt concentration, and temperature. A certain level of ionic strength (salt) is necessary for the activity of the luciferase. In the case of NaCl, at least 0.05-0.1 M of the salt is needed for a moderate rate of light emission, and about 0.5 M for the maximum light intensity. 

Heat stability The Oplophorus luminescence system is more thermostable than several other known bioluminescence systems the most stable system presently known is that of Periphylla (Section 4.5). The luminescence of the Oplophorus system is optimum at about 40°C in reference to light intensity (Fig. 3.3.3 Shimomura et al., 1978). The quantum yield of coelenterazine is nearly constant from 0°C to 20°C, decreasing slightly while the temperature is increased up to 50°C (Fig. 3.3.3) at temperatures above 50°C, the inactivation of luciferase becomes too rapid to obtain reliable data of quantum yield. In contrast, in the bioluminescence systems of Cypridina, Latia, Chaetopterus, luminous bacteria and aequorin, the relative quantum yields decrease steeply when the temperature is raised, and become almost zero at a temperature near 40-50°C (Shimomura et al., 1978). 

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. 

Mechanism of luminescence reaction. The chemical reaction of Oplophorus bioluminescence can be represented by the following simplified scheme  

The product coelenteramide is not noticeably fluorescent in aqueous solutions, but is highly fluorescent in organic solvents and also when the compound is in the hydrophobic environment of a protein. When coelenterazine is luminesced in the presence of Oplophorus luciferase, the solution after luminescence (the spent solution) is not fluorescent, presumably due to the dissociation of coelenteramide from the luciferase that provided a hydrophobic environment at the time of light emission. An analogous situation exists in the bioluminescence system of Renilla (Hori et al., 1973). 

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Anthozoa. Anthozoans are plant-shaped polyps, either solitary or colonial, completely lacking the medusoid stage. They are found along coastal waters and include the luminescent genera Renilla (the sea pansies), Cavernularia (the sea cactuses), and Ptilosarcus and Pennatula (the sea pens). Bioluminescent anthozoans emit light by a luciferin-luciferase reaction that involves coelenterazine as the... 

The scyphozoan Periphylla emits light with a luciferin-luciferase reaction using coelenterazine as the luciferin, differing from Pelagia in the same class and all luminous hydrozoans that luminesce with photoproteins. 

Quantum yield of coelenterazine. The quantum yields of coelenterazine in the luminescence reaction catalyzed by luciferases A, B and C are all close to 0.30 at 24°C, which is one of the highest values among coelenterazine luciferases. The amount of luciferase L obtained was insufficient to measure reliable data of specific activity and quantum yield. 

Coelenterazine emits chemiluminescence when dissolved in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) containing a trace amount of base. It also emits bioluminescence in aqueous media in the presence of a coelenterazine luciferase, such as Renilla luciferase or Oplophorus luciferase. In both cases, the luminescence reactions require molecular oxygen. The capability of coelenterazine to produce luminescence is attributed to the presence of the imida-zopyrazinone structure in the molecule. 

This luminous brittle star has been briefly studied recently (Mallefet and Shimomura, 2004, unpublished). The animal contained a high level of coelenterazine luciferase activity (4 x 1012 photons s-1g 1), which is comparable to those in the luminous antho-zoans such as the sea pansy Renilla and sea pen Ptilosarcus (Shimomura and Johnson, 1979b). There is no evidence for the presence of a photoprotein in this brittle star. Thus, the luminescence system of Amphiura filiformis is considered to be a coelenterazine-luciferase system, differing from that of Ophiopsila californica. The luciferase has a molecular weight of 23,000 on the basis of gel filtration on Superdex 200 Prep, and catalyzes the luminescence reaction of coelenterazine in the presence of oxygen the light emission (A.max 475 nm) is optimum at pH 7.2. 

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.

The protein contains an N-terminal signal peptide of 17 amino acid residues for secretion. The luminescence reaction of coelenterazine catalyzed by the recombinant luciferase shows a luminescence emission maximum at 485 nm, whereas the luminescence catalyzed by the native luciferase shows a maximum at 480 nm. 

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.

Renilla luciferase. The luciferase of Renilla reniformis has been purified and characterized by Karkhanis and Cormier (1971) and Matthews et al. (1977a). The purified luciferase has a molecular weight of 35,000, and catalyzes the luminescence reaction of coelenterazine. The luciferase-catalyzed luminescence is optimum at pH 7.4, at a temperature of 32°C, and in the presence of 0.5 M salt (such as NaCl or KC1). The luciferase has a specific activity of 1.8 x 1015 photons s"1mg"1, and a turnover number of 111/min. The luminescence spectrum shows a maximum at 480 nm. The absorbance A28O of a 0.1% luciferase solution is 2.1. The luciferase has a tendency to self-aggregate, forming higher molecular weight species of lower luminescence activities. 

The cDNA encoding the luciferase of Renilla reniformis has been obtained and expressed in Escherichia coli (Lorenz et al., 1991). The cDNA contained an open reading frame encoding a 314-amino acid sequence. The recombinant Renilla luciferase obtained had a molecular weight of 34,000, and showed an emission maximum at 480 nm in the luminescence reaction of coelenterazine, in good agreement with the data of natural Renilla luciferase. 

Quantum yield of luciferin. Various values of quantum yield have been reported for coelenterazine in the luminescence reaction catalyzed by Renilla luciferase 0.055 (Matthews et al., 1977a), 0.07 (Hart, et al., 1979), and 0.10-0.11 (with a recombinant form Inouye and Shimomura, 1997). The quantum yield is significantly increased in the presence of Renilla green fluorescent protein (GFP) see below. 

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.

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. 

D-Luciferin, 4 See Firefly luciferin L-Luciferin, 4 Luciferin binding protein dinoflagellates, 264, 265 Luciferin-luciferase cross-reaction, 324 Luciferins, xix-xxi, 340-342 coelenterazine, 159-179 Cypridina luciferin, 55-62, 160,... 

Glow luminescence techniques have been used extensively with luciferases as reporter genes in cell-based assays. An overview of such assays is given in Section 10.3.2 Reporter Assays below. Luciferases are enzymes which catalyze bio-luminescent reactions. Two forms are used as reporters, one originating from the firefly (firefly luciferase) and the other from Rmilla (Renilla luciferase). Due to their different origins, the enzyme structures and their respective substrates are quite different While Rmilla luciferase catalyzes the oxidation of coelenterazine, the substrate of firefly luciferase is the beetle luciferin, which is oxidized in the presence of ATP and Mg as depicted in Fig. 17. 

In efforts to understand the mechanism involved in the ATP-dependent luminescence, we investigated the conditions for the stabilization, extraction and partial purification of the active insoluble luciferase existing in the arm light organs, and then studied the characteristics of the ATP-dependent luminescence reaction of coelenterazine disulfate in the presence of the active insoluble luciferase obtained. 

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