Luminescent reaction

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. 

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. 

Conti et al. (1996) solved the crystal structure of the P. pyralis luciferase at 2.0 A resolution. The protein is folded into two compact domains, a large N-terminal portion and a small C-terminal portion. The former portion consists of a /1-barrel and two /1-sheets. The sheets are flanked by a-helices to form an aflafia five-layered structure. The C-terminal portion of the molecule forms a distinct domain, which is separated from the N-terminal domain by a wide cleft. It is suggested that the two domains will close up in the course of the luminescence reaction. 

Fig. 1.6 The time course of luminescence reaction initiated by the injection of ATP. The light intensity first rises rapidly, reaching a maximum in 0.3-0.5 sec, followed by relatively rapid decrease for the first few seconds and then a much slower decay that lasts for several minutes or more. From McElroy and Seliger, 1961, with permission from the Johns Hopkins University Press.

The optimum pH for the luminescence reaction is about 7.8, and the luminescence intensity is strongly affected by the buffer salt used... 

In the luminescence reaction of firefly luciferin (Fig. 1.12), one oxygen atom of the product CO2 is derived from the molecular oxygen while the other originates from the carboxyl group of luciferin. In the chemiluminescence reaction of an analogue of firefly luciferin in DMSO in the presence of a base, the analysis of the product CO2 has supported the dioxetanone pathway (White et al., 1975). 

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 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). 

Since then, tremendous efforts have been made to elucidate the mechanism of this complex, multi-component luminescence reaction... 

If the luciferase sample solution contains a flavin-reductase, luciferase activity can be measured by the addition of FMN and NADH, instead of FMNH2. In this case, the turnover of luciferase takes place repeatedly using the FMNH2 that is enzymatically generated thus, the luminescence reaction continues until aldehyde or NADH is exhausted. A crude luciferase extracted from luminous bacteria usually contains a flavin-reductase. 

Reduction by sodium dithionite. A small amount of sodium dithionite, solid or in solution, is added to a luciferase solution made with 50 mM phosphate, pH 7.0, containing 50 pM FMN. The amount of dithionite used should be minimal but sufficient to remove oxygen in the solution and to fully reduce the flavin. The solution made is injected into an air-equilibrated buffer solution containing a long-chain aldehyde and luciferase to initiate the luminescence reaction. With this method, the reaction mixture will be contaminated by bisulfite and bisulfate ions derived from dithionite. 

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. 

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... 

Reaction rate. The luminescence reaction of Cypridina luciferin catalyzed by Cypridina luciferase normally follows the first-order... 

Luciferase turnover. The luciferase-catalyzed light-emitting reaction that forms oxyluciferin is fast, but the hydrolysis reaction of oxyluciferin into etioluciferin by luciferase is slow. The turnover rate (catalytic center activity) of luciferase was reported to be about 30/s for the luminescence reaction, and 0.03/s for the hydrolysis of oxyluciferin (Shimomura et al., 1969). 

Side reaction. The luminescence reaction of Cypridina luciferin catalyzed by luciferase involves a side reaction (Fig. 3.1.8). In the luminescence reaction, 85-90% of luciferin is converted into oxyluciferin and CO2 accompanied by light emission, whereas 10-15% of luciferin is converted directly into etioluciferin plus a keto-acid without light emission (Shimomura and Johnson, 1971). In the chemiluminescence reactions of Cypridina luciferin in organic solvents (such as diglyme, acetone, pyridine and DMSO), the proportion of the dark side reaction... 

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. 

Structure ofF Although F has never been obtained in a completely pure state, the FAB mass spectral data of F [m/z 687 (M + Na)+ and 665 (M+H)+], and the comparison of the H and 13C NMR spectra of F with those of Oxy-F, suggested structure 6 for this compound. To confirm this structure, F was subjected to ozonolysis, followed by diazomethane treatment. The expected diester 5 was successfully isolated, indicating that 6 is indeed the structure of compound F (Nakamura et al., 1988). The structure of the luminescence reaction product of F is considered to be 8 on the basis of comparison with the dinoflagellate luminescence system (see Chapter 8). 

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... 

Quantum yield and luciferase activity The quantum yield of coelenterazine in the luminescence reaction catalyzed by Oplophorus luciferase was 0.34 when measured in 15 mM Tris-HCl buffer, pH 8.3, containing 0.05 M NaCl at 22°C (Shimomura et al., 1978). The specific activity of pure luciferase in the presence of a large excess of coelenterazine (0.9pg/ml) in the same buffer at 23°C was 1.75 x 1015 photons s 1 mg-1 (Shimomura et al., 1978). Based on these data and the molecular weight of luciferase (106,000), the turnover number of luciferase is calculated at 55/min. 

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 luminescence reaction of coelenterazine is initiated by the peroxidation of coelenterazine at its C2 carbon by molecular oxygen (Fig. 3.3.4). Then, the peroxidized coelenterazine decomposes into coelenteramide plus CO2, producing the energy needed for the light emission. For the mechanism of the decomposition of peroxide that produces the energy, two different pathways can be considered. 

The protein contains an N-terminal signal peptide of 17 amino acid residues for secretion. The luminescence reaction of coelenterazine catalyzed by the recombinant luciferase shows a luminescence emission maximum at 485 nm, whereas the luminescence catalyzed by the native luciferase shows a maximum at 480 nm. 

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