Coelenteramine 400a

Coelenteramine 400a (Coelenterazine 400a), a derivative of Coelenterazine, is a Renilla luciferase (RLuc) substrate. In the presence of Coelenteramine 400a, RLuc can emit blue light at 395 nm^[1][2]. Coelenterazine 400a will causes color change in the bioluminescence reaction of Rluc by replacing the sulfur and oxygen heteroatoms of the methylene bridge. Coelenterazine 400a provides higher signal resolution and can be used in the research of bioluminescence resonance energy transfer (BRET)^[3]. In Vitro:Luminescence reaction^[4]
1. Solution preparation:
1) Prepare 10 μL of 1.0-5.0 mM Coelenteramine 400a solution.
2) Prepare 10 μL of 2.01 mg/mL Renilla luciferase (RLuc8.6-53) solution.
3) Mix the above two solutions with 200 μL buffer.
2. Specific staining steps:
1) Record the bioluminescence spectrum of the mixed solution using a multi-channel spectrometer equipped with a liquid nitrogen-cooled CCD detector. The absolute spectral sensitivity of the spectrometer must be calibrated in advance using a 500 W spectral irradiance standard lamp.
2) A custom-made photometer equipped with a photomultiplier tube (PMT, H11890-01, Hamamatsu Photonics, Japan) was used to measure the relevant parameters of the bioluminescence reaction. The absolute response of the photometer is determined by plotting a linear graph of the relative counts measured by the photometer and the absolute counts measured by the integrating sphere.
3) In a test tube, 5-15 μL of a 10 nM solution of Coelenteramine 400a (50-150 fmol in total) is pre-placed in the photometer, and then 150 μL of a 20 μg/mL luciferase solution (in 0.1 M GTA buffer, pH 6.0, 7.0, and 8.0) is injected to start the reaction. The reaction is monitored until the luminescence reaction is complete, and the quantum yield of the bioluminescence reaction is calculated from the total number of photons obtained and the number of luciferin molecules.
4) The Michaelis constant (K_m) of the substrate Coelenteramine 400a is calculated using the Lineweaver-Burk plot. The catalytic constant (k_cat) is calculated. The fluorescence spectrum and absolute fluorescence quantum yield of the corresponding oxidation product, Coelenteramide 400a (CTMD 400a), were measured using Quantaurus-QY with the excitation light set to 300 nm. The concentration of CTMD 400a solution in DMSO was 20 μM during the measurement.