Spectra luciferase

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.

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). 

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). 

Fig. 2.4 The spectrum of bacterial luminescence measured with B. harveyi luciferase, FMN, tetradecanal and NADH, in 50 mM phosphate buffer, pH 7.0, at 0°C (dashed line from Matheson et al., 1981) and the absorption and fluorescence emission spectra of LumP (solid lines) and Rf-LumP (dotted lines) obtained from P. leiog-natbi, in 25 mM phosphate buffer, pH 7.0, containing 1 mM EDTA and 10 mM 2-mercaptoethanol, at room temperature (from Petushkov et al, 2000, with permission from Elsevier). LumP is a lumazine protein, and Rf-LumP contains riboflavin instead of lumazine in the lumazine protein. Fluorescence emission curves are at the right side of the absorption curves.

Molecular weight. The molecular weight of C. hilgendorfii luciferase reported in the past varies considerably across a range of 50,000-80,000 (Chase and Langridge, 1960 Shimomura etal., 1961, 1969 Tsuji and Sowinski, 1961 Tsuji et al., 1974) it appears most likely to be 60,000-70,000. The luciferase is an acidic protein with an isoelectric point of 4.35 (Shimomura et al., 1961). The absorption spectrum of luciferase is that of a simple protein without any prosthetic group, showing a peak at 280 nm. Absorbance value at 280 nm of a 0.1% luciferase solution is approximately 0.96 (Shimomura etal., 1969). 

Fig. 3.1.4 Bioluminescence spectrum of Cypridina luciferin catalyzed by Cypridina luciferase (A), the fluorescence excitation spectrum of oxyluciferin in the presence of luciferase (B), the fluorescence emission spectrum of the same solution as B (C), and the absorption spectrum of oxyluciferin (D). The fluorescence of oxyluciferin alone and luciferase alone are negligibly weak. Measurement conditions A, luciferin (lpg/ml) plus a trace amount of luciferase in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl B and C, oxyluciferin (20 pM) plus luciferase (0.2mg/ml) in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl D, oxyluciferin (41 pM) in 20 mM Tris-HCl buffer, pH 7.6, containing 0.2 M NaCl. All are at 20°C.

The bioluminescence reaction of Oplophorus is a typical luciferin-luciferase reaction that requires only three components luciferin (coelenterazine), luciferase and molecular oxygen. The luminescence spectrum shows a peak at about 454nm (Fig. 3.3.1). The luminescence is significantly affected by pH, salt concentration, and temperature. A certain level of ionic strength (salt) is necessary for the activity of the luciferase. In the case of NaCl, at least 0.05-0.1 M of the salt is needed for a moderate rate of light emission, and about 0.5 M for the maximum light intensity. 

Fig. 3.3.1 Luminescence spectrum of coelenterazine catalyzed by the luciferase of the decapod Oplophorus in 15 mM Tris-HCl, pH 8.3, containing 50 mM NaCl (solid line). For comparison, the luminescence catalyzed by the luciferase of the anthozoan sea pansy Renilla is shown with dashed line (in 25 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl).

Renilla luciferase. The luciferase of Renilla reniformis has been purified and characterized by Karkhanis and Cormier (1971) and Matthews et al. (1977a). The purified luciferase has a molecular weight of 35,000, and catalyzes the luminescence reaction of coelenterazine. The luciferase-catalyzed luminescence is optimum at pH 7.4, at a temperature of 32°C, and in the presence of 0.5 M salt (such as NaCl or KC1). The luciferase has a specific activity of 1.8 x 1015 photons s"1mg"1, and a turnover number of 111/min. The luminescence spectrum shows a maximum at 480 nm. The absorbance A28O of a 0.1% luciferase solution is 2.1. The luciferase has a tendency to self-aggregate, forming higher molecular weight species of lower luminescence activities. 

Fig. 6.1.5 Fluorescence spectra of the purple protein (1-4) and the luminescence spectrum measured with Latia luciferin, luciferase and the purple protein (5 Xmax 536 nm). Excitation spectra (1) and (2) were measured with emission at 630 nm and 565 nm, respectively. Emission spectra (3) and (4) were measured with excitation at 285 nm and 380 nm, respectively. From Shimomura and Johnson, 1968c, with permission from the American Chemical Society.

The light emitter in Latia luminescence. The purple protein is strongly fluorescent in red. Thus, at first glance, it would appear to be a most probable candidate for the light emitter or its precursor. However, this possibility was ruled out when we found that there is no way to relate the fluorescence of the purple protein to the bioluminescence spectrum. Thus, the luciferase must contain a chromophore that produces the light emitter. 

Latia luciferase is colorless and normally nonfluorescent. However, the luciferase fluoresces visibly in alkaline solutions. The fluorescence is most prominent in a KCN solution, showing an emission spectrum that is very close to the bioluminescence spectrum and also to the fluorescence emission of a flavin (FAD) except for the 370 nm... 

Fig. 6.1.9 Bioluminescence spectrum of Latia, and the fluorescence spectra of Latia luciferase cm 1.1) and FAD (1.5 pg/ml), both in 0.1MKCN. The fluores-...

Fig. 6.2.4 Change in the absorption spectrum of pholasin (14.5 p,M) caused by the luminescence reaction catalyzed by Pholas luciferase (1.1 p.M). The curve shown is the differential spectrum between a cell containing the mixture of pholasin and Pholas luciferase (0.9 ml in the sample light path) and two cells containing separate solutions of pholasin and the luciferase at the same concentrations (in the reference light path), all in 0.1 M Tris-HCl buffer, pH 8.5, containing 0.5 M NaCl. Four additions of ascorbate (3 iM) were made to the sample mixture to accelerate the reaction. The spectrum was recorded after 120 min with a correction for the base line. From Henry and Monny, 1977, with permission from the American Chemical Society.

Harvey (1952) demonstrated the luciferin-luciferase reaction with O. phosphorea collected at Nanaimo, British Columbia, Canada, and with O. enopla from Bermuda. McElroy (1960) partially purified the luciferin, and found that the luminescence spectrum of the luciferin-luciferase reaction of O. enopla is identical to the fluorescence spectrum of the luciferin (A.max 510 nm), and also that the luciferin is auto-oxidized by molecular oxygen without light emission. Further investigation on the bioluminescence of Odontosyllis has been made by Shimomura etal. (1963d, 1964) and Trainor (1979). Although the phenomenon is well known, the chemical structure of the luciferin and the mechanism of the luminescence reaction have not been elucidated. 

Oxyluciferin. During the luminescence reaction catalyzed by luciferase, luciferin is converted into a fluorescent compound, oxyluciferin, accompanied by the emission of greenish-blue light that spectrally matches the fluorescence of oxyluciferin (Fig. 7.2.6). The absorption spectrum of oxyluciferin is shown in Figs. 7.2.1 and 7.2.2. 

Fig. 7.2.6 (A) Luminescence spectrum of Odontosyllis luciferin-luciferase reac-...

Fig. 7.3.2 Comparison between the in vivo luminescence spectrum of a freshly exuded slime of Diplocardia longa and the in vitro luminescence spectrum measured with partially purified preparations of Diplocardia luciferin and luciferase. Reproduced from Bellisario et al., 1972, with permission from the American Chemical Society. Note that the in vitro emission maximum shifts to 490 nm when a sample of pure luciferin is used (Ohtsuka et al., 1976).

Fig. 8.4 Absorption spectrum of dinoflagellate luciferin, and the spectral changes caused by luminescence reaction after the addition of luciferase, in 0.2 M phosphate buffer, pH 6.3, containing 0.1 mM EDTA and BSA (O.lmg/ml) (Nakamura et al., 1989). Reproduced from Hastings, 1989, with permission from the American Chemical Society and John Wiley Sons Ltd.

Vervoort, J., et al. (1986). Identification of the true carbon-13 nuclear magnetic resonance spectrum of the stable intermediate II in bacterial luciferase. Biochemistry 25 8062-8067. 

Laetmogone, 301, 337 Latnpadena, 163, 339 Lampito, 216, 234, 335 Lampteroflavin, 270 Lampteromyces, 267, 270 Lampyridae, 1, 2 See also Fireflies distribution, 2 morphology, 2 Lampyris, 2, 337 Latia bioluminescence, 189 activators and inhibitors, 189 light emitter, 191 luminescence spectrum, 192 reaction scheme, 190 Latia luciferase, 183-189, 343 assay, 184... 

The dianion 117 bonded to enzyme (firefly luciferase) appears to be the emitter in blue-green firefly bioluminescence as the emission spectrum exactly matches the fluorescence of 117, and in an analogous way in the case of red bioluminescence the emitter is 115. 

The fluorescence maxima of DMOL and MMOL shift to the short-wave region upon their binding the enzyme 20 nm for DMOL and 100 nm for MMOL, and the fluorescence intensity significantly increases (Fig. 3). The observed changes in the fluorescence emission spectra of DMOL can be explained by the increase in hydrophobicity of microenvironment of the emitter on its binding with the protein. Excitation spectrum of MMOL-luciferase complex (Xem=450 nm) corresponds to the absorbance spectra of MMOL-monoanion. In buffer solution we did not seen the fluorescence spectra of this form due to the fact that in water solution electronically excited MMOL-monoanion exists in form of phenolate-ion only (it is in dianion form). The luciferase microenvironment stabilizes MMOL-monoanion by protecting its phenolic group from dissociation. It shows that in vicinity of MMOL bound to... 

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