Firefly luciferase reaction

Bioluminescence in vitro chemosensitivity assays are now used to assess the sensitivity of tumor cells (obtained by surgical or needle biopsy) to different dmgs and combinations of dmgs. Cells are grown in microwell plates in the presence of the dmgs at various concentrations. If the tumor cells are sensitive to the dmg then they do not grow, hence total extracted cellular ATP, measured using the bioluminescence firefly luciferase reaction, is low. This method has been used to optimize therapy for different soHd tumors and for leukemias (306). 

Figure 2 A more detailed scheme of ATP firefly luciferase reaction. LF = luciferase OL = oxiluciferine LFH2 = reduced luciferase PP = pirophosphate OL = oxiluciferine in the excited state.

Dukhovich A, Sillero A, Sillero MA. Time course of luciferyl adenylate synthesis in the firefly luciferase reaction. FEBS Lett 1996 395 188-90. 

In firefly luciferase reaction, the luminescence activity is enhanced by addition of Coenzyme A (CoA) and this phenomenon is explained by release of product inhibition. Also, firefly luciferase shows the sequence similarity to mammalian fatty acyl-CoA synthetase (AcCoAS) and plant 4-coumarate CoA ligase (4CL). They are classified as an adenylation enzyme for synthesizing acyl-CoA derivatives fi om carboxylic acid compounds in the presence of CoA, ATP and Mg (Scheme 1). Furthermore, it was reported that the luminescence activity of firefly luciferase is inhibited competitively by various long-chain fatty acids. We have determined that firefly luciferase is a bi-functional enzyme, catalyzing both the luminescence reaction and fatty acyl-CoA synthetic reaction. ... 

Some years ago I demonstrated that such a biochemical flash can be produced in the test tube.9,15 If oxygen is excluded from a firefly luciferase reaction mixture and then added rapidly back, a bright flash occurs, some 100 to 200 times brighter than the baseline intensity (Fig. 3). This comes from the reaction of the luciferyl adenylate active intermediate accumulated in the absence of oxygen. Note that the decay of the flash is not due to the removal of oxygen, but to the utilization of the luciferase-peroxide intermediate, so the baseline returns to a low level (Fig. 4), defined by the slow rate of reaction of ATP with luciferin. It is well known that the kinetics of firefly flashes are species specific and of functional importance in courtship communication, fixed by the rate constant for the first order decay of the peroxide intermediate formed from the adenylate. 

Fig. 3. Flashes in response to the rapid addition of oxygen to firefly luciferase reactions initiated in the complete absence of oxygen.9 A Time course of normal reaction in air. B,C,D started under strict anaerobic conditions oxygen added later at times indicated.

Fig. 14.35 Examples of various luminescent (C) The luminol reaction with horseradish reactions. (A) The firefly Luciferase reaction. peroxidase catalyst. (D) The electrolumines-(B) The bacterial luciferase reaction. cence of Ru(bpy)3.

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 ", ... 

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... 

Fig. 1.12 Mechanism of the bioluminescence reaction of firefly luciferin catalyzed by firefly luciferase. Luciferin is probably in the dianion form when bound to luciferase. Luciferase-bound luciferin is converted into an adenylate in the presence of ATP and Mg2+, splitting off pyrophosphate (PP). The adenylate is oxygenated in the presence of oxygen (air) forming a peroxide intermediate A, which forms a dioxetanone intermediate B by splitting off AMP. The decomposition of intermediate B produces the excited state of oxyluciferin monoanion (Cl) or dianion (C2). When the energy levels of the excited states fall to the ground states, Cl and C2 emit red light (Amax 615 nm) and yellow-green light (Amax 560 nm), respectively.

Orlova et al. (2003) theoretically studied the mechanism of the firefly bioluminescence reaction on the basis of the hybrid density functional theory. According to their conclusion, changes in the color of light emission by rotating the two rings on the 2-2 axis is unlikely, whereas the participation of the enol-forms of oxyluciferin in bioluminescence is plausible but not essential to explain the multicolor emission. They predicted that the color of the bioluminescence depends on the polarization of the oxyluciferin molecule (at its OH and O-termini) in the microenvironment of the luciferase active site the... 

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... 

Fontes, R., Dukhovich, A., Sillero, A., and Gunther Sillero, M. A. (1997). Synthesis of dehydroluciferin by firefly luciferase, effect of dehydrolu-ciferin, coenzyme A and nucleoside triphosphates on the luminescence reaction. Biochem. Biophys. Res. Commun. 237 445—450. 

Gates, B. J., and DeLuca, M. (1975). The production of oxyluciferin during the firefly luciferase light reaction. Arch. Biochem. Biophys. 169 616-621. 

Wannlund, J., DeLuca, M., Stempel, K., and Boyer, P. D. (1978). Use of 14C-carboxyl-luciferin in determining the mechanism of the firefly luciferase catalyzed reactions. Biochem. Biophys. Res. Commun. 81 987-992. 

Discovery of luciferin-luciferase reaction Benzoylation of Cypridina luciferin ATP requirement in firefly luminescence Requirement for long-chain aldehyde (luciferin) in bacterial luminescence... 

The colour of the light emission is yellow-green with a maximum at 560 nm. The quantum yield of the firefly luciferase bioluminescence reaction is close to 1 under optimum conditions of temperature and pH and in the presence of saturating luciferin concentration17. 

Figure 3. (a) The firefly luciferase bioluminescence reaction, (b) Structure of the specific substrate luciferin and the corresponding reaction product oxyluciferin. 

Recent applications in the field of biochemical analysis have been developed based on the highly efficient firefly luciferin-luciferase reaction, a bioluminescence reaction in which two steps can be considered ... 

Bioluminescent reactions are also employed for imaging purposes, in particular the firefly and the bacterial luciferin/luciferase ones (Fig. 1). The firefly luciferin/luciferase reaction requires ATP, magnesium ions, and oxygen. Many different luciferins and mutant luciferases have been investigated to optimize the... 

The last type of CL discussed here is bioluminescence (BL). As the term suggests, BL is an enzyme-catalyzed process found in living organisms [164, 165]. In most BL reactions, luciferin is oxidized with molecular oxygen by lucifer-ase with ATP as a cofactor. In addition, the luciferase activity depends on Ca2+ or Mg2+. The analytically most often employed system is the firefly luciferase/ D-luciferin system shown in Fig. 26. Here, ATP is necessary to form the highly energetic AMP adduct required for further reaction sequence. Subsequent cleavage... 

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