Chemical synthesis and firefly luciferase produced dehydroluciferyl- coenzyme A

Tetrahedron Letters

Volumn 45, Issue 10, 2004, Pages 2117-2120

  Fraga, Hugo ^a   Esteves Da Silva, Joaquim C G ^a   Fontes, Rui ^a  

^a UNIVERSITY OF PORTO   (Portugal)

Author keywords

Bioluminescence; Co enzyme A; Dehydroluciferyl coenzyme A; Firefly luciferase

Indexed keywords

COENZYME A; DEHYDROLUCIFERYL COENZYME A; LUCIFERASE; UNCLASSIFIED DRUG;

ARTICLE; ASSAY; BIOLUMINESCENCE; ENZYME SYNTHESIS; MASS SPECTROMETRY; REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; SPECTROPHOTOMETER; ULTRAVIOLET RADIATION;

EID: 1242317877     PISSN: 00404039     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.tetlet.2004.01.050     Document Type: Article

References (17)

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11. +). The resin was removed by filtration, and the filtrate was extracted with diethyl ether (2 × 20 mL) to remove unreacted L. The aqueous phase and the dissolved residue of the organic phase (after evaporation of the ether) were analyzed by RP-HPLC. The chromatographic system was constituted by a HP-1100 isocratic pump, a rheodyne manual injection valve, silica-based octadecyl columns and a Unicam Cristal 250 ultraviolet (UV-vis) diode array detector. The eluent was a solution of 29% methanol, 2 mM phosphate buffer (pH 7.0), and the flow rate was set to 1 mL/min. The aqueous phase was lyophilized.
12. The purification of L-CoA present in the aqueous phase was performed by HPLC using a Supelco semi-preparative chromatographic column, the eluent was a solution of 35% methanol, phosphate buffer pH 7.0, with a flow rate of 2 mL/min.
13. The reaction mixture for the enzymatic synthesis of L-CoA contained in the final volume of 200 μL: 0.5 mM CoA, 60 μM L-AMP, 100 mM Hepes buffer (pH 7.8) and luciferase (0.08 mg protein/mL). At different times of incubation (0-10 min) 35 μL aliquots were withdrawn from the reaction mixtures and added to 35 μL of 10 mM EDTA in 66% methanol. Aliquots (50 μL) were injected, the flow rate was 0.6 mL/min and the eluent was a solution of 29% methanol (v/v) and 1 mM phosphate buffer (pH 7). These elution conditions were also used with chemically synthesized L-CoA (see Fig. 4).
14. In the assays studied by fluorescence the reaction mixtures contained in the final volume of 600 μL: 50 μL of partially purified L-CoA (estimated final concentrations of L-CoA 2.5 μM, L 0.13 μM in 2 mM phosphate buffer pH 7.0), 200 μM AMP, 200 μM PPi and luciferase (0.011; 0.022; 0.033 mg protein/mL). The reactions were initiated with the addition of luciferase and were performed in quartz cell in a Perkin-Elmer luminescence spectrometer LS-50 (excitation 340 nm; emission 550 nm). In the assays studied by RP-HPLC the reaction mixtures contained in the final volume of 100 μL: 10 μL of partially purified L-CoA (estimated concentration of 140 μM L-CoA and 7 μM L); 1 mM AMP, 1 mM PPi, 100 mM Hepes (pH 7.8) and luciferase (0.13 mg protein/mL). At different times of incubation (0-60 min) 20 μL aliquots were withdrawn and added to 40 μL of 10 mM EDTA in 66% methanol. The elution conditions were the same as referred above.
15. 2 and PPase. After 5 min (synthesis from ATP, L and CoA) or 30 s (synthesis from L-AMP and CoA) of incubation, 15 μL aliquots were withdrawn from the reaction mixtures, added to 66% methanol and analyzed by RP-HPLC as referred above.
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