Luciferin luminescent oxidation

There are many kinds of luminous organisms that utilize coelenterazine as their luciferin. These organisms possess luciferases to catalyze the luminescent oxidation of coelenterazine. Coelenterazine luciferases have been isolated from about 10 kinds of organisms, including the anthozoans Renilla and Ptilosarcus, the scyphozoan... 

Shimomura, O., and Johnson, F. H. (1971). Mechanism of the luminescent oxidation of Cypridina luciferin. Biochem. Biophys. Res. Commun. 44 340-346. 

Figure 1. Luminescence reactions of Cypridina luciferin and aequorin, in which Cypridina luciferin is oxidized to oxyluciferin and etioluciferin, whereas aequorin is decomposed into coelenteramide (CLA), CO2 and Ca -bound apoaequorin. Note the structural resemblance between AF-350 and etioluciferin.

These light-producing oxidations are quite different reactions in each of the three most thoroughly studied organisms, Cypridina, bacteria, and fireflies (382,566,567,569,709,761). The structures of the two luciferins, LHk and LHjf, and of their oxidation products (oxyluciferins , 568), and the fate of the long-chain fatty aldehyde which acts as cofactor in bacterial luminescent oxidation of FMNH, are all unknown. Each luminescent system requires molecular oxj en and a potential source of electrons, in common with mixed function oxidases, but the significance of these characteristics in terms of oxidase classification remains to be determined. 

The collected results of experiments with crude and purified luciferin (99,100) indicate that it is a relatively small molecule, soluble in water, dilute acid, alkali and salt solutions, diffusible through cellophane, nonanti-genic, and not destroyed by trypsin. It has been estimated that luminescence visible to the dark-adapted eye may be obtained from luciferin in a dilution of 1 40,000,000,000 (94). With regard to some of the properties of luciferin, based on results obtained with crude extracts, it would be desirable to repeat the experiments with the purified material, inasmuch as different results are sometimes observed, e.g., cyanide apparently has no effect on the luminescent oxidation in crude extracts (91,185) but combines irreversibly with the luciferin in purified solutions (82). 

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... 

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. 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.

The most popular system in mechanistic and model studies as well as in analytical applications (clinical, food, environmental) appears to be that of firefly luciferin and luciferin-type-related model luminescence [3, 5,23, 57], The luciferase from Photinus pyralis, Photinus luciferin 4-monooxygenase (ATP-hydrolyzing), EC 1.13, 12.7, is a hydrophobic enzyme that catalyzes the air oxidation of luciferin in the presence of ATP and magnesium ions to yield light emission ... 

In chemiluminescence immunoassay the antigen is tagged with a molecule such as luminol or an acridinium ester which emits light with a high quantum yield on oxidation. Alternatively, the antigen may be labelled with a bio-luminescent molecule such as luciferin, which emits light when oxidized by the enzyme luciferase. 

Of the many types of bioluminescence in nature, that of the firefly represents the most thoroughly studied and best understood biological luminescent process. The molecular mechanism of light emission by the firefly was elucidated in the 1960s in which a dioxetanone (a-peroxy lactone) was proposed as an intermediate, formed by the luciferase-catalyzed enzymatic oxidation of the firefly luciferin with molecular oxygen (Scheme 15). This biological reaction constitutes one of the most efficient luminescent processes known to date . Hence, it is not surprising that the luciferin-luciferase system finds wide use... 

A very different light-producing reaction is used by the limpet Latia. The luciferin is an unusual terpene derivative (Fig. 23-51) that lacks any chromophore suitable for light emission.678 Evidently oxidation of this luciferin causes electronic excitation of some other molecule, presumably a "purple protein" which is also needed for luminescence. A complex of luciferin plus the purple protein is believed to react with the luciferase (abbreviated E-NH2 in Fig. 23-51). It is... 

The LIA is an immunoassay in which the antigen or antibody are labeled with either a chemiluminescent or bioluminescent tags (41, 58). Luminescent molecules are produced by oxidation reactions. Bis-phenyl oxalates in presence of hydrogen peroxides are used for chemiluminescent assays and luciferin in presence of luciferase enzyme is used for bioluminescent assays. The sensitivity of the LIA s are in the pg/ml or lower range. 

Some new luminescent and fluorescent reporters (some of them even non-substrate proteins ) are very attractive because of their easy and fast detection, explaining their current frequent use. The bacterial luciferase isolated from the Vibrio fischeri lux operon contains luxAB encoding the functional subunits and luxCDE for the synthesis and recycling of the aldehyde substrate (Prosser, 1996). Firefly (Photinus pyralis) luciferase, encoded by the luc gene catalyses the oxidative carboxylation of beetle luciferin, in which photons are emitted (LaRossa, 1998). Its short half-life and lack of any post-translational modification makes it ideal to look after effects in gene expression (Naylor, 1999). Detection of... 

Glow luminescence techniques have been used extensively with luciferases as reporter genes in cell-based assays. An overview of such assays is given in Section 10.3.2 Reporter Assays below. Luciferases are enzymes which catalyze bio-luminescent reactions. Two forms are used as reporters, one originating from the firefly (firefly luciferase) and the other from Rmilla (Renilla luciferase). Due to their different origins, the enzyme structures and their respective substrates are quite different While Rmilla luciferase catalyzes the oxidation of coelenterazine, the substrate of firefly luciferase is the beetle luciferin, which is oxidized in the presence of ATP and Mg as depicted in Fig. 17. 

Cellular ATP Determination The intensity of the bioluminescence catalyzed by luciferase was used to estimate the ATP content in bacterial cells. Bacterial samples were treated with 50% (v/v) of B-PER obtained from Pierce Biotechnology, Rockford, Il.(bacterial protein extraction reagent), a cell lysis reagent specific to bacteria. The released cellular ATP was determined from the luminescence emitted from luciferase catalyzed oxidation of luciferin. A Berthold FB12 luminometer was used to record the light output. 

The biosynthesis of luciferin (23) in the luminescent click beetle, Pyrophorus pellucens, is proposed to occur via the reaction of quinone with cysteine to give an intermediate benzothiazine, which on further reaction with cysteine gives the benzothiazole. But preliminary studies with 14C-labeled cysteine indicated, however, that an alternative fate for the first-formed thiazine, involving oxidation and ring contraction, may have to be considered.44... 

In McElroy s lab, we established that the reaction of ATP and luciferin with purified luciferase involves two steps 9 the first forms an active intermediate, later determined to be the adenylate, and the second is the reaction with oxygen, leading to an excited state and light emission. The prompt decline of luminescence over the first minutes was shown to be due to luciferase inhibition, not substrate exhaustion. All evidence indicates that the flash of the firefly is initiated by the introduction of oxygen into the photocytes, triggered by a nerve impulse, which actually does not end on the photocytes, but on adjacent cells.10 12 More recently, nitric oxide (NO)... 

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