Coelenterazine, bioluminescent

Fig. 3.3.4 Reaction mechanism of the coelenterazine bioluminescence showing two possible routes of peroxide decomposition, the dioxetanone pathway (upper route) and linear decomposition pathway (lower route). The Oplopborus bioluminescence takes place via the dioxetanone pathway. The light emitter is considered to be the amide-anion of coelenteramide (see Section 5.4).

In the case of coelenterazine (of a jeUyflsh) as well, the ancial reaction intermediate is the formation of the square-shaped unit (oxetane) as seen in Fig. 9.2. This unstable intermediate decomposes spontaneously and forms an excited state of the carbonyl group it then emits a light as it goes down to the ground state. This process requires the presence of calcium ion (Ca(II)), and hence this coelenterazine bioluminescence system is a sensitive detector of calcium. 

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). 

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.

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). 

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... 

Coelenteramide and coelenterazine. The structure of AF-350 contains the same aminopyrazine skeleton as in Cypridina etioluciferin and oxyluciferin (Fig. 3.1.8), suggesting that the bioluminescence reaction of aequorin might resemble that of Cypridina luciferin. To investigate such a possibility, we prepared the reaction product of aequorin luminescence by adding Ca2+ to a solution of aequorin. The product solution (blue fluorescent) was made acidic, and extracted with... 

Fig. 4.6.2 A scheme showing the mechanism of the Renilla bioluminescence, and the chemical structures of coelenterazine derivatives involved.

Fig. 4.6.3 Bioluminescence emission spectra measured with coelenterazine plus 1 i.M Renilla luciferase in the absence (a) and presence (b) of 1 jlM Renilla GFP. From Lorenz et al., 1991.

Soon after the hypothetical structure was published, coelenterazine was isolated as an actual substance from the liver of the luminous squid Watasenia scintillans, and it was chemically synthesized (Inoue et al., 1975). The availability of synthetic coelenterazine led to the important discovery that the treatment of the luminescence product of aequorin with coelenterazine results in the regeneration of active aequorin (Shimomura and Johnson, 1975c), which consequently confirmed the presence of a coelenterazine moiety in the aequorin molecule. During the same period, it became increasingly evident that coelenterazine is involved as a luciferin in various bioluminescent organisms, such as the sea cactus Cavernularia, the sea pen Ptilosarcus, and the sea pansy Renilla (Shimomura and Johnson, 1975b). 

The occurrence of coelenterazine in marine bioluminescent organisms is extremely widespread. Coelenterazine functions as their light-emitting substance, usually as a luciferin or the functional group of a... 

The existence of coelenterazine in various nonluminous organisms suggests that some of the coelenterazine-dependent luminous organisms might obtain coelenterazine from their food, either as the sole source of this substance or as a supplement to the coelenterazine biosynthesized in the body. In the case of the hydrozoan Aequorea aequorea, it was reported that the medusa is unable to produce its own coelenterazine and is dependent on a dietary supply of this compound for its capability of bioluminescence (Haddock et al., 2001). The organisms that biosynthesize coelenterazine remain to be identified, but it seems to be a common opinion at present that at least copepods do make their own coelenterazine. According to Thomson etal. (1995), the shrimp Systellaspis debilis is capable of coelenterazine... 

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. 

Based on the available knowledge on the chemiluminescence and bioluminescence reactions of various luciferins (firefly, Cypridina, Oplophorus and Renilla), the luminescence reaction of coelenterazine is considered to proceed as shown in Fig. 5.4 (p. 171). The reaction is initiated by the binding of O2 at the 2-position of the coelenterazine molecule, giving a peroxide. The peroxide then forms a four-membered ring dioxetanone, as in the case of the luminescence... 

The enol-sulfate form (I), which is the precursor of the luciferin in the bioluminescence system of the sea pansy Renilla (Hori et al., 1972), can be readily converted into coelenterazine by acid hydrolysis. The enol-sulfate (I), dehydrocoeienterazine (D) and the coelenterazine bound by the coelenterazine-binding proteins are important storage forms for preserving unstable coelenterazine in the bodies of luminous organisms. The disulfate form of coelenterazine (not shown in Fig. 5.5) is the luciferin in the firefly squid Watasenia (Section 6.3.1). An enol-ether form of coelenterazine bound with glucopyra-nosiduronic acid has been found in the liver of the myctophid fish Diapbus elucens (Inoue et al., 1987). 

There are many kinds of bioluminescent squids. Some of them harbor luminous bacteria for their light emission (Harvey, 1952 Haneda, 1985), but all other luminous squids currently known utilize coelenterazine or its derivatives in their bioluminescence systems, and... 

C 5. Measurements of Coelenterazine, its Derivatives, and other Important Substances in Bioluminescence... 

Chen, F.-Q., et al. (1994). Synthesis and preliminary chemi- and bioluminescence studies of a novel photolabile coelenterazine analogue with a trifluoromethyl diazirine group. Chem. Commun. 2405-2406. 

Haddock, S. H. D., Rivers, T. J., and Robison, B. H. (2001). Can coelenter-ates make coelenterazine Dietary requirement for luciferin in cnidarian bioluminescence. Proc. Natl. Acad. Sci. USA 98 11148-11151. 

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. 

Ohmiya, Y., Teranishi, K., Akutagawa, M., and Ohashi, M. (1993a). Bioluminescence activity of coelenterazine analogs after incorporation into recombinant apoaequorin. Cbem. Lett. 1993 2149-2152. 

Shimomura, O. (1987). Presence of coelenterazine in non-bioluminescent marine organisms. Comp. Biochem. Physiol. 86B 361-363. 

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