Luminescence Bioluminescence

Luminescence. Bioluminescence methods rely on the production of light by an enzyme-catalyzed reaction. The enzymatic reaction yields an excited-state product, which returns to the ground state upon emission of light, as shown in Eq. 3.23. 

Analytical Applications. Chemiluminescence and bioluminescence are useful in analysis for several reasons. (/) Modem low noise phototubes when properly instmmented can detect light fluxes as weak as 100 photons/s (1.7 x 10 eins/s). Thus luminescent reactions in which intensity depends on the concentration of a reactant of analytical interest can be used to determine attomole—2eptomole amounts (10 to 10 mol). This is especially useful for biochemical, trace metal, and pollution control analyses (93,260—266) (see Trace and residue analysis). (2) Light measurement is easily automated for routine measurements as, for example, in clinical analysis. 

Chemiluminescent labels, in which the luminescence is generated by a chemical oxidation step, and bioluminescent labels, where the energy for light emission is produced by an enzyme-substrate reaction, are additional labeling types (39,42). Luminol [521 -31 -3] CgHyN202, and acridine [260-94-6] C H N, derivatives are often used as chemiluminescent labels. 

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. 

Fig. 1.7 Spectral change of the in vitro firefly bioluminescence by pH, with Photinus pyralis luciferase in glycylglycine buffer. The normally yellow-green luminescence (Amax 560 nm) is changed into red (Xmax 615 nm) in acidic medium, accompanied by a reduction in the quantum yield. From McElroy and Seliger, 1961, with permission from Elsevier.

The sharp flash in the firefly bioluminescence reaction (Fig. 1.6) is due to the formation of a strongly inhibitory byproduct in the reaction. The inhibitor formed is dehydroluciferyl adenylate, having the structure shown below at left. In the presence of coenzyme A (CoA), however, this inhibitory adenylate is converted into dehydroluciferyl-CoA, a compound only weakly inhibitory to luminescence. Thus, an addition of CoA in the reaction medium results in a long-lasting, high level of luminescence (Airth et al., 1958 McElroy and Seliger, 1966 Ford et al., 1995 Fontes et al., 1997, 1998). 

In the lsO-incorporation experiment of Cypridina bioluminescence, the effects of the O atom exchange and contaminating CO2 are clearly seen in the relationship between the amount of luciferin luminesced and the amount of lsO atoms incorporated into the product CO2 (Fig. 1.14 Shimomura and Johnson, 1973a). The experiments were done in glycylglycine buffer, pH 7.8, the same buffer as chosen by DeLuca and Dempsey (1970). The total volume of the reaction mixture was 4 ml, with 40 ml of gas phase (see the reaction vessel in Fig. A.5 in the Appendix). The data of the luminescence reaction with 1802 gas in the H2160 medium indicates that at least 1 pmol of... 

The bioluminescence systems of Phengodidae (railroad worms) and Elateroidae (click beetles) are basically identical to that of Lampyridae (fireflies), requiring firefly luciferin, ATP, Mg2+ and a luciferase for light emission. However, there seem to be some differences. Viviani and Bechara (1995) reported that the spectra of the luminescence reactions measured with the luciferases of Brazilian fireflies (6 species) shift from the yellow-green range to the red range with lowering of the pH of the medium, like in the case of the Photinus pyralis luciferase (see Section 1.1.5), whereas the spectra... 

The order Diptera (flies) contains the glow-worms Arachnocampa and Orfelia. The bioluminescence systems of dipterans do not utilize firefly luciferin in their light-emitting reactions, differing from the bioluminescence systems of coleopterans. In dipterans, it is extremely intriguing that the bioluminescence system of Arachnocampa appears different from that of Orfelia-. the former luminescence is activated by ATP, whereas the latter luminescence is stimulated by DTT but not by ATP. 

The reported quantum yields of the long-chain aldehydes in the luminescence reaction catalyzed by P. fischeri luciferase are 0.1 for dodecanal with the standard I (Lee, 1972) 0.13 for decanal with the standard I (McCapra and Hysert, 1973) and 0.15-0.16 for decanal, dodecanal and tetradecanal with the standard III (Shimomura et al., 1972). Thus, the quantum yield of long-chain aldehydes in the bacterial bioluminescence reaction appears to be in the range of 0.10-0.16. 

The autoinducer is a low molecular weight compound that is easily leached from the cells into the culture medium. By the propagation of bacterial cells, the concentration of the autoinducer in the medium increases. When the concentration reaches a certain threshold, the biosynthesis of bioluminescence system begins, and the bacteria become luminescent. The process is also called quorum sensing (Fuqua et al., 1994). 

Inhibitors. Many common enzyme inhibitors show little or no effect on the activity of Cypridina luciferase in the luminescence reaction (Tsuji et al., 1974). However, EDTA strongly inhibits the bioluminescence reaction, showing a peculiar relationship between the... 

In our report on the bioluminescence of Meganyctiphanes (Shimomura and Johnson, 1967), the extremely unstable nature of the substance P caused us to interpret the functions of P and F incorrectly, the former as a photoprotein and the latter as a catalyst, as pointed out by Hastings (1968). The error was corrected 28 years later (Shimomura, 1995a), F being unambiguously shown to be a luciferin and P, a luciferase, on the basis that the quantum yield of F is about 0.6 at 0°C, while P can be recycled many times in the luminescence reaction. 

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. 

Heat stability The Oplophorus luminescence system is more thermostable than several other known bioluminescence systems the most stable system presently known is that of Periphylla (Section 4.5). The luminescence of the Oplophorus system is optimum at about 40°C in reference to light intensity (Fig. 3.3.3 Shimomura et al., 1978). The quantum yield of coelenterazine is nearly constant from 0°C to 20°C, decreasing slightly while the temperature is increased up to 50°C (Fig. 3.3.3) at temperatures above 50°C, the inactivation of luciferase becomes too rapid to obtain reliable data of quantum yield. In contrast, in the bioluminescence systems of Cypridina, Latia, Chaetopterus, luminous bacteria and aequorin, the relative quantum yields decrease steeply when the temperature is raised, and become almost zero at a temperature near 40-50°C (Shimomura et al., 1978). 

Mechanism of luminescence reaction. The chemical reaction of Oplophorus bioluminescence can be represented by the following simplified scheme ... 

Fig. 3.3.3 Effects of temperature on the activities of luciferase ( ) and the quantum yields of coelenterazine (o) in the Oplophorus bioluminescence reaction. The activity was measured with coelenterazine (4.5 pg) and luciferase (0.05 pg), and the quantum yields with coelenterazine (0.2 pg) and luciferase (200 pg), in 5 ml of 15 mM Tris-HC1 buffer, pH 8.3 (at 25°C), containing 50 mM NaCl. Coelenterazine was first added to the buffer solution at the designated temperature, then the luminescence reaction was started by a rapid injection of 0.1 ml of luciferase solution. Replotted from Shimomura et al., 1978, with permission from the American Chemical Society.

The product coelenteramide is not noticeably fluorescent in aqueous solutions, but is highly fluorescent in organic solvents and also when the compound is in the hydrophobic environment of a protein. When coelenterazine is luminesced in the presence of Oplophorus luciferase, the solution after luminescence (the spent solution) is not fluorescent, presumably due to the dissociation of coelenteramide from the luciferase that provided a hydrophobic environment at the time of light emission. An analogous situation exists in the bioluminescence system of Renilla (Hori et al., 1973). 

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... 

Fig. 4.1.3 Absorption spectra of aequorin (A), spent solution of aequorin after Ca2+-triggered luminescence (B), and the chromophore of aequorin (C). Fluorescence emission spectrum of the spent solution of aequorin after Ca2+-triggered bioluminescence, excited at 340 nm (D). Luminescence spectrum of aequorin triggered with Ca2+ (E). Curve C is a differential spectrum between aequorin and the protein residue (Shimomura et al., 1974b) protein concentration 0.5 mg/ml for A and B, 1.0 mg/ml for C. From Shimomura and Johnson, 1976.

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... 

Fig. 4.2.2 Left panel-. Uncorrected Ca2+-triggered bioluminescence spectrum of W92F obelin derived from O. longissima. Right panel Corrected bioluminescence spectrum of the same obelin (dotted line), and the fluorescence emission spectrum of the spent solution after luminescence (solid line). From Deng et al., 2001, with permission of the Federation of the European Biochemical Societies.

Other hydrozoans. Various bioluminescent coelenterates have been comparatively studied in reference to the components necessary for their in vivo luminescence (Morin and Hastings, 1971a Cormier et al., 1973 Shimomura and Johnson, 1975b, 1979b). 

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