Dinoflagellate luciferin

The chemical structure of dinoflagellate luciferin was found to be a tetrapyrrole, possibly derived from chlorophyll (Nakamura et al., 1989). The luciferase of G. polyedra was cloned (Bae and Hastings, 1994). Recently, the crystal structure of the third domain of the luciferase was determined (Schultz et al., 2005). 

The solution of purified dinoflagellate luciferin is yellow, showing absorption maxima at 245 and 390nm in an aqueous solution and at 241 and 388 nm in 40% acetonitrile containing 85 mM NaCl and 3 mM NaHCOs (Fig. 8.4). The compound is strongly fluorescent in blue (excitation maximum at 390 nm, emission maximum at 474 nm Fig. 8.5). The properties of this luciferin are nearly identical with those of the compound F of euphausiid shrimps (Section 3.2). The luciferin is rapidly oxidized in the presence of a trace of oxygen, and also inactivated by a weak acid, even by an acidity of pH 4 or the acidity... 

Fig. 8.4 Absorption spectrum of dinoflagellate luciferin, and the spectral changes caused by luminescence reaction after the addition of luciferase, in 0.2 M phosphate buffer, pH 6.3, containing 0.1 mM EDTA and BSA (O.lmg/ml) (Nakamura et al., 1989). Reproduced from Hastings, 1989, with permission from the American Chemical Society and John Wiley Sons Ltd.

Fig. 8.5 Corrected fluorescence spectra of partially purified dinoflagellate luciferin obtained from Dissodinium. From Njus, 1975.

Fig. 8.6 Absorption spectra of slightly autoxidized dinoflagellate luciferin in 50% ethanol (A), and the blue oxidation product in methanol (B). From Dunlap et al., 1981, with permission from the Federation of the European Biochemical Societies.

Chemical Structures of Dinoflagellate Luciferin and its Oxidation Products (Nakamura et al., 1989)... 

Mild chromic acid oxidation of luciferin (CrOs/KHSC /HiO, room temperature) yielded 3-methyl-4-vinylmaleimide (1, Fig. 8.7), 3-methyl-4-ethylmaleimide (2), and an aldehyde (3), whereas vigorous chromic acid oxidation (CrOs/2N H2SO4, 90°C) gave hema-tinic acid (4) (Dunlap et al., 1981). These results closely resemble the results of the chromic acid oxidation of the fluorescent compound F of euphausiid (p. 76), indicating a structural similarity between dinoflagellate luciferin and the compound F. 

Fig. 8.8 The chemical structures of dinoflagellate luciferin (5), the product of luminescence reaction catalyzed by luciferase (6), air-oxidation product formed at — 20°C (7), and the blue oxidation product (8). Note structural resemblance between these compounds and chlorophylls.

Fig. 8.9 Possible mechanisms of the bioluminescence reaction of dinoflagellate luciferin, based on the results of the model study (Stojanovic and Kishi, 1994b Stojanovic, 1995). The luciferin might react with molecular oxygen to form the luciferin radical cation and superoxide radical anion (A), and the latter deproto-nates the radical cation at C.132 to form (B). The collapse of the radical pair might yield the excited state of the peroxide (C). Alternatively, luciferin might be directly oxygenated to give C, and C rearranges to give the excited state of the hydrate (D) by the CIEEL mechanism. Both C and D can be the light emitter.

Storage under vacuum in a sealed tube (Method II). Substances that are extremely oxygen-sensitive, such as the fluorescent compound F of euphausiids and dinoflagellate luciferin, have to be stored in an evacuated sealed container at a low temperature. For long-term storage, they must be fuse-sealed in an evacuated glass vial using the method outlined below. 

Dunlap, J. C., Hastings, J. W., and Shimomura, O. (1981). Dinoflagellate luciferin is structurally related to chlorophyll. FEBS Lett. 135 273-276. 

Morse, D., and Mittag, M. (2000). Dinoflagellate luciferin-binding protein. Method. Enzymol. 305 258-276. 

Nakamura, H., etal. (1989). Structure of dinoflagellate luciferin and its enzymatic and nonenzymatic air-oxidation products./. Am. Chem. Soc. Ill 7607-7611. 

Stojanovic, M. N., and Kishi, Y. (1994a). Dinoflagellate bioluminescence the chromophore of dinoflagellate luciferin. Tetrahedron Lett. 35 9343-9346. 

Nakamura H, Kishi Y, Shimomura O, Morse D, Hastings JW (1989) Structure of Dinoflagellate Luciferin and Its Enzymatic and Nonenzymatic Air-Oxidation Products. J Am Chem Soc 111 7607... 

Dunlap JC, Hastings JW, Shimomura O (1981) Dinoflagellate Luciferin is Structurally Related to Chlorophyll. FEBS Lett 135 273... 

Stojanovic MN, Kishi Y (1994) Dinoflagellate Bioluminescence - The Chromophore of Dinoflagellate Luciferin. Tetrahedron Lett 35 9343... 

Figure 1 Structures of luciferins (A) limpet luciferin (e.g., Latia) (B) firefly luciferin (e.g., Photinus) (C) dinoflagellate luciferin (e.g., Gonyaulax)-, (D) coelenterate luciferin (e.g., Renilla, Aequorea) (E) bacterial luciferin (e.g., Vibrio, Photobacterium).

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