Luciferin preparation

Efforts by various investigators to demonstrate a luciferin-luciferase reaction with luminous fungi were uniformly unsuccessful until Airth and McElroy (1959) obtained a positive result. Airth s group studied further details of the luciferin-luciferase reaction using the luciferin preparation obtained from Armillaria mellea and the luciferase preparation obtained from a species identified by them... 

Preparation of Balanoglossus luciferin. The residue of the first pH 6 extraction above was re-extracted with 50 mM potassium phosphate buffer, pH 8. After centrifugation, the supernatant was used as the standard luciferin preparation. Luciferin was highly labile and easily inactivated at an extreme pH, by heat, and also by freezing and thawing. The instability resembled that of certain proteins. 

Cormier and Dure (1963) found another type of luciferin and called it protein-free luciferin. Protein-free luciferin was found in the vapor condensate of freeze-drying whole animals, and also in the 3 5-56 % ammonium sulfate fraction of the crude extract noted above. The protein-free luciferin behaved like an aromatic or heterocyclic compound and it was strongly adsorbed onto Sephadex and other chromatography media, requiring a considerable amount of solvent to elute it. The luminescence reaction of protein-free luciferin in the presence of luciferase required a 500-times higher concentration of H2O2 compared with the standard luciferin preparation. Both types of the luciferin preparation had a strong odor of iodoform. 

ATP and DPA. using the foregoing luminescent systems. Nucleotide concentrations of less than 1 x 10-9 M are easily detectable using electronic instrumentation. Firefly ludferase-luciferin preparations for ATP assays are commercially available. 

Chemiluminescence is also obtained by anionic autooxidation of (41) with oxygen ia alkaline dimethyl sulfoxide (DMSO) (216). Qc has been reported to be 10% and ketone (43) and CO2 are obtained. Several analogues of luciferin have been prepared that are also chemiluminescent when they react with oxygen ia alkaline DMSO (62). 

The live fireflies are dried over calcium chloride in a vacuum desiccator, and then their lanterns are separated by hand. An acetone powder prepared from the dried lanterns is extracted with boiling water. The cooled aqueous extract is extracted with ethyl acetate at pH 3.0, and the ethyl acetate layer is concentrated under reduced pressure. The concentrated luciferin is adsorbed on a column of Celite-Fuller s earth mixture. The column is washed with water-saturated ethyl acetate, and eluted with alkaline water at pH 8.0-8.5. The aqueous eluate of luciferin is adjusted to pH 3.0 with HCl and luciferin is... 

The luciferin-luciferase reaction of Arachnocampa was first demonstrated by Wood (1993), by mixing a cold-water extract and a cooled hot-water extract. The cold-water extract was prepared with 27 mM Tricine, pH 7.4, containing 7mM MgSC>4, 0.2 mM EDTA, 10% glycerol and 1% Triton X-100, and incubated with 1 mM ATP on ice for 18 hr. The hot-water extract was prepared by heating the cold water extract before the addition of ATP at 98°C for 5 min. The luminescence reaction was performed in the presence of 1 mM ATP. 

Extraction and purification of luciferin and luciferase (Viviani etal., 2002a) To isolate luciferin, the lanterns of the Australian A. flava were homogenized in hot 0.1 M citrate buffer, pH 5, and the mixture was heated to 95°C for 5 min. The mixture was acidified to pH 2.5-3.0 with HCl, and luciferin was extracted with ethyl acetate. Upon thin-layer chromatography (ethanol-ethyl acetate-water, 5 3 2 or 3 5 2), the active fraction of luciferin was fluorescent in purple (emission Lav 415 nm when excited at 290 nm). To isolate the luciferase, the cold-water extract prepared according to Wood (1993 see above) was chromatographed on a column of Sephacryl S-300. On the same... 

Coelenteramide and coelenterazine. The structure of AF-350 contains the same aminopyrazine skeleton as in Cypridina etioluciferin and oxyluciferin (Fig. 3.1.8), suggesting that the bioluminescence reaction of aequorin might resemble that of Cypridina luciferin. To investigate such a possibility, we prepared the reaction product of aequorin luminescence by adding Ca2+ to a solution of aequorin. The product solution (blue fluorescent) was made acidic, and extracted with... 

Luciferase-catalyzed luminescence of luciferin. Odontosyllis luciferin emits light in the presence of Mg2+, molecular oxygen and luciferase. The relationship between the luminescence intensity and the pH of the medium shows a broad optimum (Fig. 7.2.8). The luminescence reaction requires a divalent alkaline earth ion, of which Mg2+ is most effective (optimum concentration 30 mM). Monovalent cations such as Na+, K+, and NH have little effect, and many heavy metal ions, such as Hg2+, Cu2+, Co2+ and Zn2+, are generally inhibitory. The activity of crude preparations of luciferase progressively decreases by repeated dialysis and also by concentrating the solutions under reduced pressure. However, the decreased luciferase activity can be completely restored to the original activity by the addition of 1 mM HCN (added as KCN). The relationship between the concentration of HCN and the luciferase activity is shown in Fig. 7.2.9. Low concentrations of h and K3Fe(CN)6 also enhance luminescence, but their effects are only transient. 

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. 8.2 Gel filtration on a column of Sephadex G-100 at pH 8 (both panels) of the crude extract of Gonyaulax polyedra cells prepared at pH 8 (upper panel) and prepared at pH 6 (lower panel). The activities of the 35 kDa and 130 kDa luciferases are measured by the addition of an excess of luciferin at pH 6.3 ( ) or at pH 8(A). The activity of the luciferin-bound LBP (luciferin-binding protein) in the upper panel is measured after the addition of an excess of 35 kDa luciferase at pH 6.3 ( ). In the lower panel, the LBP activity can be obtained by the addition of an excess of luciferin at pH 8, followed by the removal of unbound luciferin with a small column of Sephadex G-25 before the luminescence assay of bound luciferin at pH 6.3 (see the Section 8.2.8). The Overlap in the upper panel is the light emission resulting from the mixing of an aliquot of the fractions with pH 6.3 buffer. From Fogel and Hastings, 1971, with permission from Elsevier.

Molecular characteristics of luciferase. A molecule of the luciferase of G. polyedra comprises three homologous domains (Li et al., 1997 Li and Hastings, 1998). The full-length luciferase (135 kDa) and each of the individual domains are most active at pH 6.3, and they show very little activity at pH 8.0. Morishita et al. (2002) prepared a recombinant Pyrocystis lunula luciferase consisting of mainly the third domain. This recombinant enzyme catalyzed the light emission of luciferin (luminescence A.max 474 nm) and the enzyme was active at pH 8.0. The recombinant enzyme of the third domain of G. polyedra luciferase was crystallized and its X-ray structure was determined (Schultz et al., 2005). A -barrel pocket putatively for substrate binding and catalysis was identified in the structure, and... 

The preparations of luciferin (Ln, an electron acceptor) and soluble enzyme used were crude or only partially purified. The luciferase was an insoluble particulate material, possibly composed of many substances having various functions. Moreover, the luciferin-luciferase reaction was negative when both luciferin and luciferase were prepared from certain species of luminous fungus. It appears that the light production reported was the result of a complex mechanism involving unknown substances in the test mixture, and probably the crucial step of the light-emitting reaction is not represented by the above schemes. 

According to Dure and Cormier (1961, 1963) and Cormier and Dure (1963), they made the preparations of luciferase and luciferin from Balanoglossus biminiensis, collected on Sapelo Island, Georgia, and investigated the luciferin-luciferase reaction, as summarized below. 

Structure determination of luciferin. Once a luciferin is obtained in a sufficient purity, the determination of luciferin structure should be attempted most of the important properties of luciferin are usually already obtained during the course of purification as a necessity. The structural study is considerably more straightforward than the extraction and purification, due to the availability of advanced methods, such as high-resolution mass spectrometry and various NMR techniques. If help or collaboration is needed in structure determination, the attractiveness of a luciferin will make it easy to find a good collaborator. However, the purified luciferin is usually an extremely precious material considering the effort spent in preparing it. To avoid accidental loss of the purified material, the chosen collaborator must have solid knowledge and experience in structure determination a criterion to be considered is that the person has successfully done the structure determination of at least one new natural product. 

Bitler, B., and McElroy, W. D. (1957). The preparation and properties of crystalline firefly luciferin. Arch. Biochem. Biophys. 72 358-368. 

Usami, K., and Isobe, M. (1996). Chemiluminescent characters of hydroperoxide and dioxetanone of coelenterate luciferin analog prepared by low-temperature photooxygenation. Chem. Lett. 3 215-216. 

Synthetic luciferin as well as purified preparations of native and recombinant firefly luciferases are now commercially available allowing the bioluminescent determination of ATP to be used as a routine analysis technique in some laboratories. 

Prepare a stock solution of D-luciferin at 15 mg/ml concentration in DPBS. Filter sterilize through a 0.2 pm filter. Prepare enough to inject 10 pl/g of body weight. Each mouse should receive 150 mg D-luciferin/kg body weight. For example, for a 30 g mouse, inject 300 pi of 15 mg/ml stock to deliver 4.5 mg of luciferin (see Note 5). 

Nigaki alcohol (18) has been identified by spectroscopic and chemical means as a constituent of Picrasma ailanthoides Planchon. Latia luciferin (19) has been synthesized in a stereoselective manner. A key step in this synthesis involves the addition of lithium dimethylcuprate to an enol phosphate derived from a 8-keto-ester to form an a,/3-unsaturated ester. Dehydro-/8-ionilideneacetic acid (20), an important intermediate in the synthesis of abscisic acid, has been prepared, as have the two nor-abscisic acid derivatives (21). The metabolite (22) of abscisic acid has been identified in the seeds of Robinia pseudacacia... 

The formation of aldehydes from 1,1-disubstituted epoxides has occasionally found use in synthesis, although simpler aldehydes in particular tend to form dioxolane dimers by BFs-induced reaction with epoxide. Hill et a converted the epoxide (94), which had been prepared from a 3-ionone derivative, into luciferin aldehyde (95) by treatment with cold BF3 etherate (equation 38). 

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