D-Luciferin

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. 

Wada, N. (1989). The active oxygen participating in the primary process of firefly D-(—)-luciferin chemiexcitation in dimethylsulfoxide. Toyo Daigaku Kogakubu Kenkyu Hokoku 25 27-34. 

D-Luciferin, 4 See Firefly luciferin L-Luciferin, 4 Luciferin binding protein dinoflagellates, 264, 265 Luciferin-luciferase cross-reaction, 324 Luciferins, xix-xxi, 340-342 coelenterazine, 159-179 Cypridina luciferin, 55-62, 160,... 

ATP-dependent oxidation of D-luciferin with firefly luciferase ... 

The last type of CL discussed here is bioluminescence (BL). As the term suggests, BL is an enzyme-catalyzed process found in living organisms [164, 165]. In most BL reactions, luciferin is oxidized with molecular oxygen by lucifer-ase with ATP as a cofactor. In addition, the luciferase activity depends on Ca2+ or Mg2+. The analytically most often employed system is the firefly luciferase/ D-luciferin system shown in Fig. 26. Here, ATP is necessary to form the highly energetic AMP adduct required for further reaction sequence. Subsequent cleavage... 

Fig. 26 Mechanism of the ATP- and Mg2+-dependent firefly luciferase catalyzed bioluminescence oxidation reaction of D-luciferin (d-LH2) to oxyluciferin (oxy-L)...

Low-/c t GTPase in tubulin polymerization, MICROTUBULE ASSEMBLY KINETICS LUCIFERASE D(-)-Luciferin,... 

Luciferin D-luciferin powder (beetle luciferin potassium salt, Promega, Madison, WI, USA) resuspended 25 mg/mL in water for injection (Galenica Senese). 

Evaluation of Substrate d -Luciferin Distribution by In Vivo Imaging Time Course... 

Any semi-quantitative evaluation of photon emission derived from the luciferin/luciferase enzymatic reaction by in vivo imaging requires prior study of the diffusion to all organs of the enzyme substrate, D-luciferin, at a concentration sufficient to saturate the reporter enzymatic activity (4). Indeed the diffusion of the substrate may change depending on the strain of mouse used or the specific formulation of the substrate. Here follows the protocol we adopted to this aim ... 

Five reporter mice are anesthetized using a subcutaneous (s.c.) injection of 50 piL ketamine-xylazine solution or by gas anesthesia exposing mice for 2 min to isofluorane. They are then injected intraperitoneal (i.p.) with 80 mg/kg D-luciferin. 

Zhang Y, Bressler JP, Neal J, Lai B, Bhang HE, Laterra J, Pomper MG. (2007) ABCG2/BCRP expression modulates D-luciferin based bioluminescence imaging. Cancer Res. 67(19), 9389-97. 

D-luciferin Firefly, potassium salt, 1.0 g/vial (Xenogen/ Caliper Life Sciences, Hopkinton, MA). 

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). 

Inject animals by an intraperitoneal i.p. route with D-luciferin solution (150 mg/kg, in DPBS). Subsequently, place animals into the plexiglass anesthesia box (XGI-8, Gas Anesthesia system connected to the IVIS system) and set the vaporizer dial to 2.5-3.5% isoflurane. 

At 12 min (see Note 6) after D-luciferin injection, image animals in the dorsal position (laying on back) for 2-5 s at field of view (FOV) 23 (E), with medium binning selected in the IVIS menu (see Note 7). 

Twenty-four hours after the last in vivo bioluminescence imaging, inject the animals i.p. with 150 mg/kg D-luciferin and euthanize them 2 min later. 

Expose the abdominal cavity through a midline incision and locate para-aortic lymph nodes (Fig. 14.1B). Subsequently, surgically remove the lymph nodes and place them in DPBS containing 300 ptg/ml D-luciferin in 96-well polypropylene plates. 

In vivo luciferase imaging Escain (Mylan Inc., Tokyo, Japan), D-luciferin and In vivo luciferase imaging system (IVIS) (Caliper Life Sciences, MA). 

The mice were anaesthetized with Escain and i.p. injected with D-luciferin (150 mg/kg). 

Luciferin buffer 35 pM D-luciferin (Roche Diagnostics), 60 mM DTT (Sigma-Aldrich), 10 mM magnesium sulfate, 1 mM ATP, in 25 mM glycyl-glycin-NaOH buffer, pH 7.8. 

The reporter gene is the luc operon from the firefly Photinus pyralis. This operon codes for an enzyme, luciferase, that catalyses the reaction between D-luciferin (the substrate), and oxygen, and requires ATP as a cofactor. This reaction produces the emission light that provides the measure of enzyme activity. The oxidation of one molecule of luciferin involves one ATP molecule and releases one photon (a stoichiometric reaction) (DeLuca and McElroy, 1974 Wood el al. 1989). The reaction can be summarized as follows ... 

The specific activities of luciferase and its mutants were determined with a luminometer AB-2100 (Atto, Tokyo, Japan) and 96-well white microplate (Nalge-Nunc, Tokyo, Japan). The reaction mixture contained 100 mM Tricine, 10 mM MgS04, 300 jtiM D-luciferin (LH2), 10 mM Na-ATP, and 1 mg/ml BSA, pH 8.0 unless otherwise indicated. Enzyme concentrations used were 0.1 nM for WT, 10 nM for K529A, and 1 /xM for the N-domain. The specific activity was determined by a peak-height-based assay based on the maximum intensity for the N-domain, and the peak intensity within the first 10 s for WT and K529A, respectively. 

Wada N, Sameshima K. Ab initio calculation for D-(-)-luciferin and its intermediates in dimethyl sulfoxide. Bioluminescence Chemiluminescence, Proceedings of the International Symposium, 11th, 2001 Singapore World Scientific Publishing Co. Ltd., 251-4. 

---