27-Luciferin

It has been suggested that the oxidation of Photinus pyralis luciferin (170) forms oxyluciferin (171) as the oxidized product (Scheme 88) (405-407), which was too unstable to be isolated and was rapidly converted in three other compounds. 

Later, fireflv oxyluciferin was successfully synthesi2ed (403. 408) and has been isolated and identified in firefly lanterns (luciola cruaciata) after the lanterns were treated with pyridine and acetic anhydride to prevent decomposition (409). In 1972, Suzuki and Goto firmly established that oxyluciferin is involved in the bioluminescence of firefly lanterns and in the chemiluminescence of firefly luciferin (403. 410).. A. mechanism involving a four-membered ring cyclic peroxide has been proposed for the reaction (406. 411). However, it was not confirmed by 0 -labelinE experiments (412). 

Firefly. Firefly luciferase (EC 1.13.12.7) is a homodimeric enzyme (62 kDa subunit) that has binding sites for firefly luciferin and Mg ATP . Amino acid sequence analysis has iadicated that beetle luciferases evolved from coen2yme A synthetase (206). Firefly bioluminescence is the most efficient bioluminescent reaction known, with Qc reported to be 88% (5), and at 562 nm (56). At low pH and ia the presence of certain metal ions (eg, Pb ", ... 

Luciferin (41) (R = H) reacts with adenosiae triphosphate (ATP) and the en2yme to give a complex (42) of the adenylate ester (41) R =... 

Chemiluminescence is also obtained by anionic autooxidation of (41) with oxygen ia alkaline dimethyl sulfoxide (DMSO) (216). Qc has been reported to be 10% and ketone (43) and CO2 are obtained. Several analogues of luciferin have been prepared that are also chemiluminescent when they react with oxygen ia alkaline DMSO (62). 

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. 

A widely prescribed drug for the treatment of gastric ulcers with the generic name cimetidine is a synthetic imidazole derivative. Firefly luciferin is a thiazole derivative that is the naturally occuning light-emitting substance present in fireflies. 

Firefly luciferin is an exanple of an azole that contains a benzene ring fused to the five-rnernbered ring. Such structures are fairiy common. Another example is benzimidazole, present as a structural unit in vitfflnin B12. Some compounds related to benzimidazole include purine and its fflnino-substituted derivative adenine, one of the so-called heterocyclic bases found in DNA and RNA (Chapter 28). 

The fireflies, railroad worms, and click beetles use the same luciferin in their luminescence reactions. Recent studies on the railroad worms and the click beetles have greatly contributed to the biochemical understanding of the firefly bioluminescence (see Section 1.2). Concerning luminous Diptera, significant progress has been made only recently. 

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 following schemes represent the overall reaction of firefly bioluminescence (McElroy and DeLuca, 1978), where E is luciferase LH2 is D-luciferin PP is pyrophosphate AMP is adenosine phosphate LH2-AMP is D-luciferyl adenylate (an anhydride formed between the carboxyl group of luciferin and the phosphate group of AMP) and L is oxyluciferin. 

In the first step, luciferin is converted into luciferyl adenylate by ATP in the presence of Mg2+. In the second step, luciferyl adenylate is oxidized by molecular oxygen resulting in the emission of yellow-green light, of which the mechanism is discussed in Sections 1.1.6 and 1.1.7. Both steps, (1) and (2), are catalyzed by luciferase. The reaction of the first step is slower than that of the second step, thus the first step is the rate-limiting step. 

The live fireflies are dried over calcium chloride in a vacuum desiccator, and then their lanterns are separated by hand. An acetone powder prepared from the dried lanterns is extracted with boiling water. The cooled aqueous extract is extracted with ethyl acetate at pH 3.0, and the ethyl acetate layer is concentrated under reduced pressure. The concentrated luciferin is adsorbed on a column of Celite-Fuller s earth mixture. The column is washed with water-saturated ethyl acetate, and eluted with alkaline water at pH 8.0-8.5. The aqueous eluate of luciferin is adjusted to pH 3.0 with HCl and luciferin is... 

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. 1.3 Absorption spectra of firefly luciferin at pH 7.0 or below (solid line, Xmax 327-328 nm) and at pH higher than 9.0 (dashed line, Amax 381-384 nm). Reproduced from McElroy and Seliger, 1961, with permission from the Johns Hopkins University Press.

Fig. 1.4 Absorption spectrum of a spent luminescence solution of firefly luciferin containing luciferase-oxyluciferin after dialysis in 0.1 M potassium phosphate, pH 7.8. Replotted from the data of Gates and DeLuca, 1975, with permission from Elsevier.

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.

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