Luciferases

Bioluminescence can also be used as the basis for immunoassay. For example, bacterial luciferase has been used in a co-immobilized system to detect and quantify progesterone using a competitive immunoassay format (34), and other luciferase-based immunoassays have been used to quantify insulin, digoxin, biotin, and other clinically important analytes (35). 

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 ", ... 

Genes lux) encoding luciferase and the other proteias iavolved ia the bioluminescent reaction have been cloned. PuxA. and luxP genes code for the luciferase subunits, and the fatty acid reductase complex is coded by the luxCDE genes. 

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. 

As httle as lO " g of ATP can be detected with carefiiUy purified luciferase. Commercial luciferase contains enough residual ATP to cause background emission and increase the detection limit to 10 g (294). The method has been used to determine bacterial concentrations in water. As few as lO" cells/mL of Lscherichia coli, which contains as Httle as 10 g of ATP per cell, can be detected (294). Numerous species of bacteria have been studied using this technique (293—295). 

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). 

Although most anesthetics are achiral or are adininistered as racemic mixture, the anesthetic actions are stereoselective. This property can define a specific, rather than a nonspecific, site of action. Stereoselectivity is observed for such barbiturates as thiopental, pentobarbital, and secobarbital. The (3)-enantiomer is modestly more potent (56,57). Additionally, the volatile anesthetic isoflurane also shows stereoselectivity. The (3)-enantiomer is the more active (58). Further evidence that proteins might serve as appropriate targets for general anesthetics come from observations that anesthetics inhibit the activity of the enzyme luciferase. The potencies parallel the anesthetic activities closely (59,60). 

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. 

Log-dose response curves, 17 Lowest positive Log(DR-l) value, 105 LTD4, 191 Luciferase, 83, 88... 

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. 

In the first step, luciferin is converted into luciferyl adenylate by ATP in the presence of Mg2+. In the second step, luciferyl adenylate is oxidized by molecular oxygen resulting in the emission of yellow-green light, of which the mechanism is discussed in Sections 1.1.6 and 1.1.7. Both steps, (1) and (2), are catalyzed by luciferase. The reaction of the first step is slower than that of the second step, thus the first step is the rate-limiting step. 

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. 

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. 1.7 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.

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