Luciferin-luciferase Luminescence Reaction

The luciferin-luciferase reaction of Arachnocampa was first demonstrated by Wood (1993), by mixing a cold-water extract and a cooled hot-water extract. The cold-water extract was prepared with 27 mM Tricine, pH 7.4, containing 7mM MgSC>4, 0.2 mM EDTA, 10% glycerol and 1% Triton X-100, and incubated with 1 mM ATP on ice for 18 hr. The hot-water extract was prepared by heating the cold water extract before the addition of ATP at 98°C for 5 min. The luminescence reaction was performed in the presence of 1 mM ATP. 

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. 

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. 

The New Zealand freshwater limpet Latia neritoides (Fig. 6.1.1) is the only known example of a freshwater luminous organism, with two possible exceptions certain species of luminous bacteria and the larvae of certain species of fireflies. The limpet inhabits shallow clear streams in the North Island of New Zealand, clinging to stones and rocks. Latia has a small oval-shaped shell (6-8 mm long), and secretes a luminous mucus that emits a greenish glow around the body only when disturbed the limpet does not show a spontaneous luminescence. The luminescence of Latia was first reported by Suter (1890) and further details including a positive luciferin-luciferase reaction were described by Bowden (1950). Both the luciferin and the luciferase have... 

Harvey (1917) noted that the fresh arm photophores of Watasenia scintillans do not show a luciferin-luciferase reaction, and Shoji (1919) using a gas chamber of purified hydrogen demonstrated that molecular oxygen is needed for the luminescence. 

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. 7.2.8 Influence of pH on the luminescence intensity of the Odontosyllis luciferin-luciferase reaction at room temperature. From Shimomura et ai, 1963d, with permission from John Wiley Sc Sons Ltd.

Regarding the polychaete Tomopteris, the author has briefly examined its bioluminescence using a few specimens donated by Dr. Steven Haddock. The specimens did not show a luciferin-luciferase reaction, as noted by Harvey (1952). The luminescence could be... 

Fig. 8.1 Luminescence spectra of luciferin-luciferase reaction of the dinoflagellate Gonyaulax polyedra (Lingulodinium polyedrum) in a solution (solid line), isolated scintillons (x), and living Gonyaulax cells (o). From Hastings et al., 1966.

Chemiluminescent compounds and their precursors in P. stipticus. Although P. stipticus is negative in the luciferin-luciferase reaction, crude extracts of this fungus are chemiluminescent, like the luciferin obtained from Ompbalia flavida by Kuwabara and Wassink (1966). The chemiluminescence is elicited by the addition of H2O2 and Fe2+ under a mild condition of pH 5-8, and the luminescence is strongly... 

The role of a cationic surfactant must be to provide a necessary hydrophobic and polarized environment for the molecule of luciferin for its luminescence reaction. In the case of a common luciferin-luciferase reaction, such an environment is provided by the enzyme luciferase. The chemical structures of PMs, as well as that of the natural luciferin, have not been determined yet (see the next section). 

Dure and Cormier (1961) demonstrated a luciferin-luciferase reaction for the first time in the extracts of the acorn worm Balanoglossus biminiensis, and also discovered that the luminescence reaction is stimulated by H2O2, of which the details are described below. Recently, Kanakubo and Isobe (2005) reported the chemical structure of a probable luciferin of another acorn worm Ptychodera flava. 

Haneda, Y., and Johnson, F. H. (1958). The luciferin-luciferase reaction in a fish, Parapriacanthus beryciformis, of newly discovered luminescence. Proc. Natl. Acad. Sci. USA 44 127-129. 

Discovery of luciferin-luciferase reaction Benzoylation of Cypridina luciferin ATP requirement in firefly luminescence Requirement for long-chain aldehyde (luciferin) in bacterial luminescence... 

Each of 10 pL of aequorin and luciferase solution was added to a microtiter plate, and 100 pL of 50 mM Ca " in 50 mM HEPES-KOH buffer (pH 7.0) was added, and then the bioluminescent intensity was integrated for 1 s by a MicroLumat LB96P luminescent reader (EG G Berthold, Germany), immediately. Then, 100 pL of the bioluminescence reagent for luciferase (containing 40 mM ATP, 1.4 mM luciferin, 300 mM MgS04 in 50 mM HEPES-KOH buffer, pH 7.0) was added to the same wells. The bioluminescent intensity from luciferin-luciferase reaction was integrated for 1 s after a delay of 2 s. 

There are a number of techniques available to extract ATP from cells. Some use trichloroacetic acid (TCA), which also aids in stabilizing the molecule. TCA has a good extraction capacity, is relatively quick, has a reduced potential for human error, and does not discriminate between the different microbiological fractions of activated sludge. Luminescence produced in the luciferin-luciferase reaction can be measured using scintillation counting equipment or a range of luminometers currently on the market. 

The emission of light from fireflies has enchanted observers for millennia. However, it was not until comparatively recently (1947) that adenosine triphosphate (ATP) was identified as being the key component in the enzymatically controlled luciferin luminescence. The analytical utility of firefly bioluminescence was first demonstrated in 1952. Since that time the luciferin-luciferase reaction has been extensively employed for analysis in a wide variety of scientific disciplines. 

Luminescence reaction (Viviani et al., 2002a) The luciferin-luciferase luminescence reaction was carried out in 0.1 M Tris-HCl, pH 8.0, containing 2mM ATP and 4mM Mg2+. Mixing luciferase with luciferin and ATP resulted in an emission of light with rapid onset and a kinetically complex decay. Further additions of fresh luciferase, after the luminescence has decayed to about 10% of its maximum value, resulted in additional luminescence responses similar to the initial one (Fig. 1.15). According to the authors, the repetitive light emission occurred in consequence of the inhibition of luciferase by a reaction product, as seen in the case of the firefly system (McElroy et al., 1953). The luminescence spectrum showed a peak at 487nm (Fig. 1.16). 

Fig. 3.1.8 A diagram showing the reactions involved in the luciferase-catalyzed luminescence of Cypridina luciferin.

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