Oplophorus Luciferase

Extraction and purification (Shimomura et al., 1978). Dr. Yata Haneda and Dr. Shoji Inoue kindly provided us with specimens of O. gracilirostris, which had been picked out one by one from large commercial catches of Sergestes lucens netted in Suruga Bay, Japan. The material was air-dried or freeze-dried before shipment to the U.S. 

Dried shrimp was ground, defatted with benzene, and then extracted with cold water. The luciferase extracted was purified first by a batch adsorption onto DEAE cellulose (elution with 0.4 M NaCl), followed by gel filtration on a column of Sephadex G-150, anion-exchange chromatography on a column of DEAE-cellulose (gradient elution 0.05-0.5 M NaCl), and gel filtration on a column of Ultrogel AcA 34. The specific activity of the purified luciferase was 1.7 x 1015 photons s 1 mg-1, and the yield in terms of luciferase activity was about 28%. 

The protein can be further purified by hydrophobic interaction chromatography on a column of Butyl Sepharose 4 Fast Flow (Pharmacia elution with decreasing concentration of (NH4)2S04 starting at 1.5 M), and gel filtration on a column of Superdex 200 Prep (Pharmacia Inouye et al., 2000). 

The native luciferase having a molecular weight of 106,000 probably consists of two units of the functional 19 kDa protein and two units of the 35 kDa protein. The value of A28o,icm for a solution containing 1 mg/ml of the native luciferase is calculated to be about 0.9 from the inferred amino acid sequence. The function of the 35 kDa protein remains unclear, although it might have a role in the stabilization of the 19 kDa protein. 

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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. 

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). 

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. 

Inouye, S., and Shimomura, O. (1997). The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate. Biochem. Biophys. Res. Commun. 233 349-353. 

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. 

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).

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). 

Fig. 3.3.2 Influence of pH on the activity of luciferase ( ) and the quantum yield of coelenterazine (o) in the bioluminescence of Oplophorus. The measurements were made with coelenterazine (4.5 pg) and luciferase (0.02 pg) for the former, and coelenterazine (0.1 pg) and luciferase (100 pg) for the latter, in 5 ml of 10 mM buffer solutions at 24° C. The buffer solutions used sodium acetate (pH 5.0), sodium phosphate (pH 6.0-7.5), Tris-HCl (pH 7.5-9.1), and sodium carbonate (pH 9.5-10.5), all containing 50 mM NaCl. Replotted from Shimomura et al., 1978, with permission from the American Chemical Society.

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.

Effect of pH. The light emission from most bioluminescence systems is affected by the pH of the medium, and some luciferases and photoproteins can be made inactive at certain pH ranges without resulting in permanent inactivation. For example, the luminescence of euphausiids can be quenched at pH 6, the luminescence of aequorin can be suppressed at pH 4.2-4A, and the luciferase of the decapod shrimp Oplophorus becomes inactive at about pH 4. In the case of Cypridina luminescence, however, the acidification of an extract to below pH 5 results in an irreversible inactivation of the luciferase. 

Inouye, S., Watanabe, K., Nakamura, H., and Shimomura, O. (2000). Secretional luciferase of the luminous shrimp Oplophorus gracilirostris cDNA cloning of a novel imidazopyrazinone luciferase. FEBS Lett. 481 19-25. 

Nakamura, H., etal. (1997). Efficient bioluminescence of bisdeoxycoelenter-azine with the luciferase of a deep-sea shrimp Oplophorus. Tetrahedron Lett. 38 6405-6406. 

Studies with Cypridina (154), Oplophorus 150) and Renilla 155) luciferases have uncovered the following steps (Scheme 26) 156). First, the appropriate luciferase deprotonates the luciferin (62) to its tetrahedral carbanion (64). Reaction of the latter with oxygen yields the hydroperoxide (65). Cyclization affords the so-called high energy intermediate, the dioxetanone (66), which on cleavage loses carbon dioxide to give the amide (67) accompanied by chemiluminescence. 

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