Luciferin phosphate

Bioluminescence has been used in immunoassays (Terouanne et al., 1986) but its complexity and high background, arising from ATP contamination, thus far make this system less attractive. Biolumines-cent assays have also been proposed with alkaline phosphatase using luciferin phosphate as the substrate (Hauber and Geiger, 1987) and... 

Another interesting and potentially promising approach is using luciferin phosphate (Fig. 4d) as a substrate for phosphatases... 

Another variant of PP2A assay is the one reported by Isobe et al. [166] where a firefly bioluminescence system is used for the detection of protein phosphatase 2A inhibitors, in which luciferin phosphate is hydrolyzed to luciferin and inorganic phosphate by protein phosphatase 2A. The recent commercial availability of the phosphatase enzymes, which obviates the need to isolate them from animal tissues, also makes this approach very attractive. However, not all microcystins variants react with protein phosphatase enzymes to a similar extent [161,163] and the assay is sensitive to protein phosphatase inhibitors other than microcystins, such as okadaic acid, tautomycin, and calyculin A. In addition, the cyanobacterial sample itself may contain phosphatase activity that masks the presence of toxins [160]. As a consequence, the lack of specificity of the protein phosphatase inhibition assays requires that additional confirmatory analytical methods be employed for specific analysis of cyanobacterial toxins. 

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. 

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. 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.

Fig. 3.1.5 Effects of salt concentration on the activity of Cypridina luciferase (solid lines) and quantum yield (dotted lines). In the activity measurement, Cypridina luciferin (1 pg/ml) was luminesced with a trace amount of luciferase in 2.5 mM HEPES buffer, pH 7.5, containing a salt to be tested, at 20°C. In the measurement of quantum yield, luciferin (1 pg/ml) was luminesced with luciferase (20 pg/ml) in 20 mM sodium phosphate buffer (for the NaCl data) or MES buffer (for the CaCl2 data), pH 6.7.

Fig. 3.1.7 Effects of temperature on the activity of Cypridina luciferase (solid line) and the quantum yield of Cypridina luciferin (dashed line). Luciferin (1 pg/ml) was luminesced in the presence of luciferase (a trace amount for the activity measurement 20 pg/ml for the quantum yield) in 50 mM sodium phosphate buffer, pH 6.8, containing 0.1 M NaCl.

Johnson et al. (1962) measured the quantum yield of Cypridina luciferin in the luciferase-catalyzed reaction for the first time, using a photomultiplier calibrated with two kinds of standard lamps. The measurement gave a value of 0.28 0.04 at 4°C in 50 mM sodium phosphate buffer, pH 6.5, containing 0.3 M NaCl. The quantum yield... 

Assay of luciferin and luciferase. To assay luciferin, 1 ml of 5 mM phosphate buffer, pH 6.8, containing crude luciferase (which contains the purple protein) is added to 5—l0 xl of an ethanolic solution of luciferin, and the total light emitted is measured. To assay the activity of luciferase in crude material, 1 ml of 5 mM phosphate buffer, pH 6.8, containing a standard amount of luciferin is added to 5-10 xl of the sample, and the intensity of emitted light is measured. To assay purified luciferase, 1 ml of 5 mM phosphate buffer, pH 6.8, containing a standard amount of luciferin and a standard amount of purple protein is added to 5-10 xl of a sample, and the light intensity is measured (Shimomura and Johnson, 1968b,c). 

Fig. 6.1.6 Effect of the purple protein on the luminescence of Latia luciferin (0.16 jxg) plus Latia luciferase (A280,icm 1.2, 10 pi) in 5 ml of 5mM sodium phosphate buffer, pH 6.8. The amounts of the purple protein solution ( 280,1 cm 0.6) used 20 pi (curve 1), 5 pi (curve 2), 1 pi (curve 3), 0.5 pi (curve 4), 0.2 pi (curve 5), and none (curve 6). From Shimomura and Johnson, 1968c, with permission from the American Chemical Society.

Fig. 7.3.3 Relationship between the concentration of H2O2 and the peak intensity of luminescence, when 0.2 ml of a H2O2 solution was injected into a mixture of 0.575 ml of 0.1 M potassium phosphate (pH 7.5), 0.025 ml of a solution of Diplocardia luciferin, and 0.2 ml of luciferase solution (0.12 mg). 1 LU = 109 quanta/s. From Bellisario et al., 1972, with permission from the American Chemical Society.

Fig. 7.3.4 Kinetic profiles of the Diplocardia bioluminescence reaction, when Diplocardia luciferase, H2O2, or Diplocardia luciferin was injected last. In each case, 0.1 ml of the last component was injected into 0.9 ml of the mixture of other components, to give the final concentrations Diplocardia luciferase, 0.1 unit/ml Diplocardia luciferin, 32 mM and H2O2, 32 mM, in 0.1 M potassium phosphate buffer, pH 7.5. From Rudie et al., 1981, with permission from the American Chemical Society.

Assay of the activities of luciferin and luciferase. Small volumes (5-50 xl) of luciferase and luciferin (Section 8.2.4) are mixed in 2 ml of 0.2 M citrate buffer, pH 6.3, or 0.2 M phosphate buffer, pH 8. The measurement is made in terms of the total light emission or the initial maximum light intensity that is reached within a few seconds. The... 

The reddish yellow solution is diluted with 4-5 volumes of cold water containing 5 mM 2-mercaptoethanol to reduce the conductivity to 0.7 m 2 1 or less, and applied to a column of DEAE-cellulose (coarse grade 5 x 15 cm) equilibrated with 2mM potassium phosphate, pH 8.0, containing 5mM 2-mercaptoethanol. The column is first washed with the cold equilibration buffer, then luciferin is eluted with a linear increase of potassium phosphate from 2 mM to 0.3 M, monitoring the effluent by fluorescence and the absorption at 390 nm. The rest of the purification method described below is adapted from the... 

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.

To assay the amount of LBP, first an excess amount of luciferin is added to the sample at pH 8 to saturate the binding site of LBP, and then the excess luciferin is removed by gel filtration using a small column of Sephadex G-25 (about 1 ml volume) also at pH 8. The luciferin-bound LBP is eluted at the void volume. To measure the amount of LBP, the following assay buffer is added to a small portion of the elu-ate 0.2 M phosphate, pH 6.3, containing 0.25 mM EDTA, 0.1 mg/ml of BSA, and luciferase (Morse and Mittag, 2000). The total light obtained represents a relative amount of LBP the absolute amount (the weight or the number of molecules) cannot be obtained because the quantum yield of the luminescence reaction is not known. 

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. 

Fig. 10.4.2 The effects of temperature (left panel) and pH (right panel) on the peak intensities of the Balanoglossus luminescence reaction. In the measurements of the temperature effect, 0.5 ml of 0.176 mM H2O2 was injected into a mixture of 1.2 ml of 0.5 M Tris buffer (pH 8.2), 0.3 ml of luciferase, and 1 ml of luciferin, at various temperatures. For the pH effect, the Tris buffer (pH 8.2) was replaced with the Tris buffers and phosphate buffers that have various pH values, and the measurements were made at room temperature. From Dure and Cormier, 1963, with permission from the American Society for Biochemistry and Molecular Biology.

ATP Luciferin-luciferase Phosphate buffer (pH 7.8) End-column CL detection 5 nM 84... 

The same reaction was recently proposed to detect creatine kinase (CK), an enzyme of high clinical significance in relation to the investigation of skeletal muscle disease and the diagnosis of myocardial infarct or cerebrovascular accidents. As ATP is a reaction product obtained from the reaction of ADP with creatine phosphate catalyzed by CK, this enzyme can be indirectly measured by the CL intensity read from the subsequent reaction of ATP with luciferin. Using the technique of electrophoretically mediated microanalysis (EMMA), it is possible to detect the enzyme using nanoliter volumes of biological sample with an improved speed and simplicity with respect to a conventional colorimetric method [100],... 

Ito, K. Nakagawa, K. Murakami, S. Arakawa, H. Maeda, M. Highly sensitive simultaneous bioluminescent measurement of acetate kinase and pyruvate phosphate dikinase activities using a firefly luciferase-luciferin reaction and its application to a tandem bioluminescent enzyme immunoassay. Anal. Sci., 19, 105-109 (2003)... 

The structure of firefly luciferin has been confirmed by total synthesis. The firefly emits a ycllow-grccn luminescence, and luciferin in this case is a benzthiazole derivative. Activation of the firefly luciferin involves the elimination of pyrophosphate from ATP widi the formation of an add anhydride linkage between the carboxyl group of luciferin and the phosphate group of adenylic acid forming luciferyl-adenylate. 

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... 

Fig. 6.5. Time-course of ATP synthesis in a suspension of PPase, ATPase liposomes. 25 /nl liposomes (about 0.07 mg protein per ml) were suspended in a medium containing 1 ml 0.2 M glycylglycine (pH 7.8), 0.2 ml luciferin/luciferase assay, 20 fjl 100 mM sodium phosphate, 10 pi 10 mM ADP and 50 pi 10 mM sodium pyrophosphate. The final concentration of MgClj was about 10 mM. At the arrow, 25 pi liposomes were added. The resulting luminescence was measured in an LKB luminometer 1250. The light output was calibrated by addition of a known amount of ATP. (From Ref. 98.)...

One widely used enzyme mediated chemiluminescence immunoassay uses the firefly enzyme luciferase that catalyzes the oxidation of o-Luciferin in the presence of ATP. o-luciferin, but not luciferin esters such as phosphates, is oxidized in the presence... 

Fig. 5 Structures of luminogenic phosphatase substrates (a) CDP-star (b) o-luciferin 6 -0-phosphate...

The problem of a poor detection limit was caused by high background ATP and by the low sensitivity of the luciferin-luciferase (L-L) reagent. We have already developed an ATP elimination system using two ATP degrading enzymes (adenosine phosphate deaminase and apyrase) and a surfactant tolerant luciferase that was a mutated Luciola lateralis firefly luciferase. We optimized this elimination system, and investigated its suitability as a detection system. 

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