Luciferin fluorescence

Goelenterate. Coelenterates Penilla reformis (sea pansy) -cradViequoreaforskalea (jelly fish) produce bioluminescence by similar processes (223). The basic luciferin stmcture is (49) (224) and excited amide (50) is the emitter. The stmctural relationship to Varela is evident. A stmctural analogue where R = CH is active ia bioluminescence. The quantum yield is about 4% (223), with at 509 nm (56). This reaction iavolves a charge transfer between green fluorescent proteia and the excited-state coelenterate oxylucifetin. 

Properties of luciferin. The crystals are microscopic needles, which melt with decomposition at 205-210°C (Bitler and McElroy, 1957). It is a quite stable luciferin compared with some other luciferins, such as Cypridina luciferin and the luciferins of krill and dinoflagellates. It is not significantly affected by lOmM H2SO4 and lOmM NaOH at room temperature in air. The absorption spectral data of luciferin are shown in Fig. 1.3 (McElroy and Seliger, 1961). The molar absorption coefficient of the 328 nm peak in acidic solutions and that of the 384 nm peak in basic solutions are both 18,200 (Morton et al., 1969). Luciferin is fluorescent, showing an emission maximum at 537 nm in both acidic and basic conditions, although the intensity of the fluorescence is lower in acidic solution than in basic solution (fluorescence quantum yields 0.62 in basic condition, and 0.25 in acidic condition Morton et al., 1969). The chemical synthesis... 

Fig. 1.5 Fluorescence emission spectrum of the luciferase-oxyluciferin complex in the same solution as in Fig. 1.4 (solid line), compared with the luminescence spectrum of firefly luciferin measured in glycylglycine buffer, pH 7.6 (dotted line). The former curve from Gates and DeLuca, 1975 the latter from Selinger and McElroy, 1960, both with permission from Elsevier.

Extraction and purification of luciferin and luciferase (Viviani etal., 2002a) To isolate luciferin, the lanterns of the Australian A. flava were homogenized in hot 0.1 M citrate buffer, pH 5, and the mixture was heated to 95°C for 5 min. The mixture was acidified to pH 2.5-3.0 with HCl, and luciferin was extracted with ethyl acetate. Upon thin-layer chromatography (ethanol-ethyl acetate-water, 5 3 2 or 3 5 2), the active fraction of luciferin was fluorescent in purple (emission Lav 415 nm when excited at 290 nm). To isolate the luciferase, the cold-water extract prepared according to Wood (1993 see above) was chromatographed on a column of Sephacryl S-300. On the same... 

Fig. 3.1.4 Bioluminescence spectrum of Cypridina luciferin catalyzed by Cypridina luciferase (A), the fluorescence excitation spectrum of oxyluciferin in the presence of luciferase (B), the fluorescence emission spectrum of the same solution as B (C), and the absorption spectrum of oxyluciferin (D). The fluorescence of oxyluciferin alone and luciferase alone are negligibly weak. Measurement conditions A, luciferin (lpg/ml) plus a trace amount of luciferase in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl B and C, oxyluciferin (20 pM) plus luciferase (0.2mg/ml) in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl D, oxyluciferin (41 pM) in 20 mM Tris-HCl buffer, pH 7.6, containing 0.2 M NaCl. All are at 20°C.

The fluorescent compound F, a luciferin, emits blue light (Amax 476 nm Fig. 3.2.4) in the presence of molecular oxygen and the protein P, a luciferase. In the luminescence reaction, F is changed into an oxidized form (structure 8, Fig. 3.2.6). The luminescence reaction is highly sensitive to pH, with a narrow optimal range around pH 7.8 (Fig. 3.2.2) the optimum salt concentration is 0.15 M for NaCl... 

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... 

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. 6.1.5 Fluorescence spectra of the purple protein (1-4) and the luminescence spectrum measured with Latia luciferin, luciferase and the purple protein (5 Xmax 536 nm). Excitation spectra (1) and (2) were measured with emission at 630 nm and 565 nm, respectively. Emission spectra (3) and (4) were measured with excitation at 285 nm and 380 nm, respectively. From Shimomura and Johnson, 1968c, with permission from the American Chemical Society.

The absorption spectra of Watasenia luciferin (coelenterazine disulfate) and Watasenia oxyluciferin (coelenteramide disulfate) are shown in Fig. 6.3.2. Watasenia luciferin in neutral aqueous solutions is auto-oxidized in air more rapidly than coelenterazine, and the compound emits a strong chemiluminescence in the presence of H2O2 ( 10mM) plus Fe2+ ( 0.2mM). Watasenia oxyluciferin is strongly fluorescent in aqueous solutions (Amax 400 nm), 500 times stronger than the fluorescence of coelenteramide in aqueous media (Goto et al., 1974). 

Harvey (1952) demonstrated the luciferin-luciferase reaction with O. phosphorea collected at Nanaimo, British Columbia, Canada, and with O. enopla from Bermuda. McElroy (1960) partially purified the luciferin, and found that the luminescence spectrum of the luciferin-luciferase reaction of O. enopla is identical to the fluorescence spectrum of the luciferin (A.max 510 nm), and also that the luciferin is auto-oxidized by molecular oxygen without light emission. Further investigation on the bioluminescence of Odontosyllis has been made by Shimomura etal. (1963d, 1964) and Trainor (1979). Although the phenomenon is well known, the chemical structure of the luciferin and the mechanism of the luminescence reaction have not been elucidated. 

Fig. 7.2.3 Spectral changes of Odontosyllis luciferin caused by various reagents (Shimomura et ai, 1963d). The peak wavelengths (nm) of the absorption, luminescence and fluorescence spectra are shown in parentheses.

Oxyluciferin. During the luminescence reaction catalyzed by luciferase, luciferin is converted into a fluorescent compound, oxyluciferin, accompanied by the emission of greenish-blue light that spectrally matches the fluorescence of oxyluciferin (Fig. 7.2.6). The absorption spectrum of oxyluciferin is shown in Figs. 7.2.1 and 7.2.2. 

The reddish yellow solution is diluted with 4-5 volumes of cold water containing 5 mM 2-mercaptoethanol to reduce the conductivity to 0.7 m 2 1 or less, and applied to a column of DEAE-cellulose (coarse grade 5 x 15 cm) equilibrated with 2mM potassium phosphate, pH 8.0, containing 5mM 2-mercaptoethanol. The column is first washed with the cold equilibration buffer, then luciferin is eluted with a linear increase of potassium phosphate from 2 mM to 0.3 M, monitoring the effluent by fluorescence and the absorption at 390 nm. The rest of the purification method described below is adapted from the... 

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

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. 

The luciferin showed an absorption spectrum identical with that of the fluorescent substance F of euphausiid (Xmax 388 nm), and gave... 

According to the Kuwabara-Wassink paper, the purified luciferin in aqueous neutral buffer solution showed an absorption maximum at 320 nm, and a fluorescence emission peak at 490 nm. The luminescence emission maximum measured with Airth s fungal luciferase system was 524 nm at pH 6.5, whereas the chemiluminescence emission maximum of the luciferin with H2O2 plus a droplet of strong NaOH plus ferrous sulfate was 542 nm. No information was reported on the chemical nature of the luciferin. 

Fig. 9.13 Absorption spectrum of one of the luciferin precursors of Mycena cit-ricolor in methanol (dash-dot line, A.max 369 nm). The absorption and fluorescence emission spectra of the decylamine-activation product of the same precursor in neutral aqueous solution (solid lines abs. Amax 372 nm and fl. Xmax 460 nm), and in ethanol (broken lines abs. Amax 375 nm and fl. Amax 522 nm). The chemiluminescence spectrum of the same activation product (dotted line, A.max 580 nm). The dotted line (7max 320 nm) is the absorption spectrum of M. citricolor natural luciferin reported by Kuwabara and Wassink (1966).

Properties of the activation product. The two decylamine-activation products (luciferins) showed similar absorption characteristics (A.max 372 nm in water, and 375 nm in ethanol), which clearly differ from the absorption peak of the natural luciferin (320 nm) reported by Kuwabara and Wassink (1966). The fluorescence emission of the activation products varied significantly by solvents, showing a peak at 460 nm in neutral aqueous solution and a broad peak at 485-522 nm in ethanol. They emitted chemiluminescence (A.max 580 nm) in the presence of CTAB, H2O2 and Fe2+ (Fig. 9.13), in resemblance to the (NH4)2S04-activation product of panal (A.max 570 nm). 

Fig. 10.6.2 The pempherid fish Parapriacanthus ransonneti dissected to show the pyloric caeca and portions of the visceral organs, photographed by daylight (top) and by ultraviolet light (bottom). The strong yellowish fluorescence in ultraviolet light is due to the presence of a large amount of Cypridina luciferin in the pyloric caeca and portions of the intestine. From Haneda and Johnson, 1962.

Name of Luciferin Molecular Formula (Mr) Absorption Max. (nm) (e value) Fluorescence Emission Max. (nm) Luminescence Max. (nm) Quantum Yield... 

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. 

G andelman, O. A., Brovko, L. U., Ugarova, N. N., and Shchegolev, A. A. (1990). The bioluminescence system of firefly. A fluorescence spectroscopy study of the interaction of the reaction product, oxy-luciferin, and its analogs with luciferase. Biokhimiya 55 1052-1058. 

Hart, R. C., Matthews, J. C., Hori, K., and Cormier, M. J. (1979). Renilla reniformis bioluminescence Luciferase-catalyzed production of nonradiating excited states from luciferin analogues and elucidation of the excited state species involved in energy transfer to Renilla green fluorescent protein. Biochemistry 18 2204-2210. 

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