Luciferase system

According to the Kuwabara-Wassink paper, the purified luciferin in aqueous neutral buffer solution showed an absorption maximum at 320 nm, and a fluorescence emission peak at 490 nm. The luminescence emission maximum measured with Airth s fungal luciferase system was 524 nm at pH 6.5, whereas the chemiluminescence emission maximum of the luciferin with H2O2 plus a droplet of strong NaOH plus ferrous sulfate was 542 nm. No information was reported on the chemical nature of the luciferin. 

Despite the relatively common occurrence of luminous echino-derms, only two species of ophiuroid, Ophiopsila califomica and Amphiura filiformis, have been biochemically investigated. It is surprising that the former species luminesces with a photoprotein system, whereas the latter emits light with a luciferin-luciferase system. 

This luminous brittle star has been briefly studied recently (Mallefet and Shimomura, 2004, unpublished). The animal contained a high level of coelenterazine luciferase activity (4 x 1012 photons s-1g 1), which is comparable to those in the luminous antho-zoans such as the sea pansy Renilla and sea pen Ptilosarcus (Shimomura and Johnson, 1979b). There is no evidence for the presence of a photoprotein in this brittle star. Thus, the luminescence system of Amphiura filiformis is considered to be a coelenterazine-luciferase system, differing from that of Ophiopsila californica. The luciferase has a molecular weight of 23,000 on the basis of gel filtration on Superdex 200 Prep, and catalyzes the luminescence reaction of coelenterazine in the presence of oxygen the light emission (A.max 475 nm) is optimum at pH 7.2. 

The first step of a chemical study should be the quantitative measurements of coelenterazine, dehydrocoelenterazine, and a coelenter-azine-specific luciferase, in the light organs, liver, digestive tract (with empty stomach), and eggs if available (see Section C5 of Appendix for the method). A clear, unequivocal presence of a coelenterazine luciferase indicates the involvement of a luciferin-luciferase system,... 

Kamzolkina, O. V., Bekker, Z. E., and Egorov, N. S. (1984). Extraction of the luciferin-luciferase system from the fungus Armillariella mellea. Biologicheskie Nauki (Moscow) 1984 73-77. 

Oba, Y., et al. (2004). Identification of the luciferin-luciferase system and quantification of coelenterazine by mass spectrometry in the deep-see luminous ostracod Conchoecia pseudodiscophora. ChemBioChem 5 1495-1499. 

Enzymatic Assay. The enzymatic (luciferase) assay for adenosine triphosphate (ATP) is one of the methods applied to areas of biocidal control in oil production operation [1454]. A reliable method for the determination of ATP is the measurement of bioluminescence produced by the luciferin luciferase system. 

Brovko L.Y., Gandelman O.A., Polenova T.E., Ugarova N.N. Kinetics of bioluminescence in the firefly luciferin-luciferase system. Biochemistry (Russia) 1994 59(2) 195-201. 

A rapid, nondestructive method based on determination of the spatial distribution of ATP, as a potential bioindicator of microbial presence and activity on monuments, artworks, and other samples related to the cultural heritage, was developed [57], After cell lysis, ATP was detected using the bioluminescent firefly luciferin-luciferase system and the method was tested on different kinds of surfaces and matrices. Figure 3 reports the localization of biodeteriogen agents on a marble specimen. Sample geometry is a critical point especially when a quantitative analysis has to be performed however, the developed method showed that with opti-... 

Figure 3 Localization of biodeteriogen agents on a marble sample by luminescent ATP detection using the firefly luciferin-luciferase system. (Courtesy of Dr. G. Ranalli, University of Molise, Campobasso, Italy.)...

Of the many types of bioluminescence in nature, that of the firefly represents the most thoroughly studied and best understood biological luminescent process. The molecular mechanism of light emission by the firefly was elucidated in the 1960s in which a dioxetanone (a-peroxy lactone) was proposed as an intermediate, formed by the luciferase-catalyzed enzymatic oxidation of the firefly luciferin with molecular oxygen (Scheme 15). This biological reaction constitutes one of the most efficient luminescent processes known to date . Hence, it is not surprising that the luciferin-luciferase system finds wide use... 

It must be pointed out that, despite the lack of direct experimental support for this hypothesis, the involvement of the electron transfer in luminol chemiexcitation is related to the chemiexcitation steps proposed for several highly efficient chemi- and biolu-minescent systems, such as activated peroxyoxalate chemiluminescence and the firefly luciferin/luciferase system (Section... 

Analogously to the firefly luciferin/luciferase system, the general chemiluminescence mechanism postulated for 9-carboxyacridinium derivatives proposes the 1,2-dioxetanone 45 as high-energy intermediate However, this 1,2-dioxetanone is the only intermediate that has not yet been isolated . The cleavage of the peroxidic ring presumably results in the release of CO2 and the formation of an acridan residue in its electronically excited state (Scheme 32). 

The jellyfish Aequorea contains a photoprotein, which emits light only when calcium ions are present.672 673 Since light emission can be measured with great sensitivity (modern photomultipliers can be used to count light quanta) the protein aequorin and related photoproteins674a are used as a sensitive indicator of calcium ion concentration.674 (In a similar way the firefly luciferin-luciferase system, which requires ATP for activation, is widely used in an assay for ATP.)... 

The flow of leukocytes was studied in square capillaries fabricated on a Si chip, and sealed with a PDMS or Pyrex cover plate. This capillary size (cross section of 4 pm2) is similar to the diameter of a human blood capillary, but is less than both the average diameter of a leukocyte cell (10 pm) and its nucleus (6 pm). Figure 8.32 shows the difference in the flow behavior of two leukocytes (possibly neutrophils) [1175]. Deformation-induced release of ATP from erythrocytes in PDMS channels was studied. The released ATP was detected by chemiluminescence using the luciferin/luciferase system [169]. 

Synthesis of a luciferin. Latia luciferin (4), the specific substrate for the luciferase system of l.citi nerit aides, has been synthesized in three steps from dihydro-/3-iononc (I). Reaction of (I) with dimethyloxosulfonium methylide, both... 

Bioluminescence is a unique type of chemiluminescence found in biological systems these reactions can be classified as either pyridine- or adenine-nucleotide linked systems or enzyme-substrate systems. In clinical enzymology, the most commonly used system is the firefly luciferin-luciferase system for the measurement of ATP ... 

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