Luciferases firefly

In BLI, the target eells of interest (e.g. tumor or baeteria) are first transfected with die luciferase (firefly) gene that is incorporated into the cell s genome and replicated upon cell division. When the substrate luciferase is then administered to the tumor or pathogen-bearing animal, the bioluminescence reactions take place ... 

A visual selection system A visual identification enables the selection of transformed cells, such as Gus, lacZ (p-galactosidase), luc and lux (luciferase, firefly Photinus pyralis, and Vibrio harveyi, respectively), gfp (jellyfish Aequo-rea victoria), and AtMYB12 (the transcriptional activator in Arabidopsis thali-ana that is indispensable for the biosynthesis of fiavonols). 

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 ", ... 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

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). 

The membrane enzyme luciferase, responsible for light emission in fireflies, is sensitive to anesthetics (20,21), and the concentrations of inhalational agents which inhibit luciferase are the same as those which cause general anesthesia. Studies of various classes of inhalational agents and luciferase demonstrated that above a certain chain length in a homologous series, a point is reached where higher members are not anesthetic. The same cut-off effect in efficacy is observed in anesthesia (22). This effect is not explainable by Hpid theory. 

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. 

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.

The apparent molecular weights of both natural P. pyralis luciferase and an active luciferase obtained from P. pyralis by the in vitro RNA translation were 62,000 by SDS-PAGE (Wood et al., 1984), in contrast to the value of 100,000 that had been widely referred to in the field for almost 30 years. Luciferases from other species of firefly probably have similar molecular weights. Presently, the molecular masses of firefly luciferases are considered to be 60-62 kDa. 

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.

In the postulated bioluminescence mechanism, firefly luciferin is adenylated in the presence of luciferase, ATP and Mg2+. Luciferyl adenylate in the active site of luciferase is quickly oxygenated at its tertiary carbon (position 4), forming a hydroperoxide intermediate (A). 

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.

Bioluminescence of firefly luciferin can produce a wide range of colors when catalyzed by different luciferases obtained from various species of fireflies, with their emission maxima ranging from 535 nm (yellow-green) to 638 nm (red). Apparently, each spectrum is emitted from a single emitting species they are not the composites of the yellow-green peak and the red peak (Seliger and McElroy, 1964). 

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 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... 

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). 

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... 

Fig. 6.3.5 A reaction scheme proposed by Tsuji (2002) for the Watasenia bioluminescence. The proposed mechanism involves the adenylation of luciferase-bound luciferin by ATP, like in the bioluminescence of fireflies. However, the AMP group is split off from luciferin before the oxygenation of luciferin, differing from the mechanism of the firefly bioluminescence. Thus the role of ATP in the Watasenia bioluminescence reaction remains unclear. Reproduced with permission from Elsevier.

When H2O2 is a necessary component of a luminescence system, it can be removed by catalase. If a luminescence system involves superoxide anion, the light emission can be quenched by destroying O2 with superoxide dismutase (SOD). The ATP cofactor usually present in the fresh extracts of the fireflies and the millipede Luminodesmus can be used up by their spontaneous luminescence reactions, eventually resulting in dark (nonluminous) extracts containing a luciferase or photoprotein. The process is, however, accompanied by a corresponding loss in the amount of luciferin or photoprotein. The use of ATPase and the elimination of Mg2+ in the extract may prevent such a loss. 

Baldwin, T. O. (1996). Firefly luciferase the structure is known, but the mystery remains. Structure 4 223-228. 

Branchini, B. R., and Rollins, C. B. (1989). High-performance liquid chromatography-based purification of firefly luciferases. Photochem. Photobiol. 50 679-684. 

Branchini, B. R., et al. (1997). Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent. J. Biol. Cbem. 272 19359-19364. 

Branchini, B. R., etal. (2005). Mutagenesis evidence that the partial reactions of firefly bioluminescence are catalyzed by different conformations of the luciferase C-terminal domain. Biochemistry 44 1385-1393. 

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