Luciferin luciferase and

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.

Luminescence reaction (Bellisario et al., 1972). Mixing the luciferin, luciferase and H2O2 results in an emission of light, regardless of the presence or absence of molecular oxygen. The in vitro luminescence with partially purified luciferin and luciferase (A.max 503 nm) was spectrally similar to the in vivo luminescence from freshly exuded slime (A.max 507 nm) (Fig. 7.3.2). However, Ohtsuka et al. (1976) reported that the emission maximum was found at 490 nm when a pure sample of luciferin was used. 

The present book describes all the significant studies and findings on the chemistry of the more than 30 different bioluminescent systems presently known, accompanied by over 1000 selected references. It includes descriptions of the purification and properties of bioluminescent compounds, such as luciferins, luciferases and photoproteins, and the mechanisms of luminescence reactions. To make the book more useful than a mere review volume and to save researchers time in looking into original references, I have included a considerable amount of original experimental methods, data and graphs. In addition, I have included some new data and experimental methods unavailable elsewhere. I hope this volume will be useful to researchers and students, and it will be my greatest pleasure if this book contributes... 

The methods of the isolation and purification of various luciferins, luciferases and photoproteins are described in detail as much as possible because of their importance. There have been considerable changes in the methods, techniques and materials used during the 50-year span covered by this book. However, the underlying principles of the purification methods have not changed significantly the old methods and techniques are often very useful for the present research when the principles involved are understood. 

The luciferin-luciferase reaction of fireflies was first demonstrated by Harvey (1917), although the light observed was weak and short-lasting. Thirty years after Harvey s discovery, McElroy (1947) made a crucial breakthrough in the study of firefly bioluminescence. He found that the light-emitting reaction requires ATP as a cofactor. The addition of ATP to the mixtures of luciferin and luciferase... 

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. 

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). 

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. 

Bivalvia. The bivalve Pholas is historically important because the concept of luciferin-luciferase reaction was established with this clam (Dubois, 1887). It is the only bivalve that is well known and biochemically investigated. The details of the Pholas bioluminescence are given in Section 6.2. 

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... 

Kojima et al. (2000a) that the purified luciferase exhibited a luciferin-luciferase reaction with Latia luciferin without any cofactor. Nevertheless, the purple protein is a conspicuous presence in the live organisms and it is highly likely that it enhances the bioluminescence of Latia in nature. 

On the basis of the luciferin-luciferase reaction discovered by Dubois (1887), Michelson, Henry and their associates studied the biochemistry of the Pholas bioluminescence for several years beginning in 1970 (Michelson, 1978). They isolated, purified, and characterized the luciferin and the luciferase, and published about a dozen papers in which the luciferin isolated was referred to as Pholas luciferin. Since the luciferin is clearly a protein, later authors called it pholasin following the traditional way of naming a photoprotein... 

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. 7.2.9 Influence of cyanide and iodine on the Odontosyllis luciferin-luciferase luminescence reaction. Luciferin solution (0.1 ml) was first mixed with a HCN solution (0.1ml), and then the mixture was injected into 8 ml of 20 mM magnesium acetate containing luciferase. The concentrations of HCN shown in the figure are the final concentrations. In the control experiment, HCN was omitted. In the experiment labeled added at 0.5 min, 0.1 ml of HCN solution was injected to the control mixture 0.5 min after the start of the luminescence reaction to give a final concentration of 0.1 mM HCN. Arrows indicate the injection of a solution of I2-3KI to the control mixture to give a final concentration of 0.1 mM I2. From Shimomura et al., 1963d, with permission from John Wiley Sons Ltd.

Assays of earthworm luciferin and luciferase (Bellisario et al., 1972). The standard assay mixture contains luciferin, luciferase... 

When an excess amount of luciferin was preincubated with H2O2 before the addition of luciferase, the quantum yields of luciferase and the luciferin-H202 adduct were found to be 0.63 and 0.03, respectively (Rudie et al., 1981). The aldehyde group of luciferin is probably converted into the corresponding acid in the luminescence reaction, although it has not been experimentally confirmed. 

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.

Assay of the activities of luciferin and luciferase. Small volumes (5-50 xl) of luciferase and luciferin (Section 8.2.4) are mixed in 2 ml of 0.2 M citrate buffer, pH 6.3, or 0.2 M phosphate buffer, pH 8. The measurement is made in terms of the total light emission or the initial maximum light intensity that is reached within a few seconds. The... 

Luciferin binding protein (LBP) binds luciferin at pH 8 but not at pH 6 (Fogel and Hastings, 1971) thus, LBP inhibits the luciferin-luciferase reaction at pH 8 but not at pH 6. Luciferin bound to LBP is stable, differing from the free form of luciferin that is extremely unstable. The molecular size of the Gonyaulax LBP was considered... 

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