Quantum yield luciferase firefly

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 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. 

Firefly luciferase catalyzes the reaction of adenosine triphosphate and a substituted benzothiazole luciferin. The quantum yield of the reaction is high and therefore measurement of the enzyme offers good sensitivity. Coupling of luciferase to other molecules is a problem because there is a highly reactive amino group in the active site that must be protected from reaction with coupling rei ents (36, 37). 

Bacterial L. has not yet been characterized. The corresponding luciferase produces luminescence in the presence of FMNH and straight chain aldehydes with more than 7 C-atoms. Structural elucidation of L. was delayed on account of their low natural concentration. 30,000 fireflies were required for the isolation of IS mg L., and 40,000 sea pansies yielded only O.S mg of the Renitla L. Many L. and synthetic analogs show a spontaneous luminescence in proton-free solvents, such as dimethyl sulfoxide, but the quantum yield is lower than in bioluminescence. See also Photoproteins. 

Two types of bioluminescence can be characterized firefly-QL, and bacterial-BL,. BL of living systems is extremely bright because each molecule of the product is formed in an electronically-excited state. The quantum yield of BL reactions is extremely high (0.1-1.0) compared to the CL of non-enzymatie oxidized organic matter. Hence, high efficiency is reached by the application of highly specific enzymes - luciferases. These are especially effective for their substrates — luciferins, which is one of the main conditions needed for chemical redox energy transformations into... 

The luciferase from the North American firefly P. pyralis is a monomeric enzyme (62 kDa) consisting of 550 amino acid residues. The firefly luciferase produces light by the ATP-dependent oxidation of D(-)-luciferin (LH2) (see Scheme 8.3). The reaction involves an enzyme-hound luciferyl adenylate intermediate. The peak light emission occurs at 562 nm (yellow—green, quantum yield of 0.88) in dianionic form between pH 7 and 8 or at 610 nm (red, quantum yield of 0.2) in monoanionic form at pH values below 7. The red shift also occurs in the presence of Zn (2.3 mM) or Cd (12 mM) (58). 

The reaction has fairly strict solvent limitations. Polar aprotic solvents are required, the most usual being the dialkyl phthalates and ethylene glycol dimethyl ether. Other esters are also useful, but acetone, ethanol and halogenated hydrocarbons are much less effective. Some admixture of tert. butanol (up to about 5%) has little effect, but water and other alcohols reduce the quantum yield. It is interesting that the active site of firefly luciferase is known to be particularly hydrophobic. The mechanisms of the two reactions are rather similar in that both require a highly active ester (the adenylate in the case of the firefly). Attack by peroxide occurs in both cases (m mmolecular in the luciferin) and this process may require a non-aqueous environment for maximum efficiency. Some of the most efficient oxalates are listed in Table 2. 

---