Coelenteramine 400a (Coelenterazine 400a), a Coelenterazine derivative, serves as a substrate for Renilla luciferase (RLuc), facilitating the emission of blue light at 395 nm [1] [2]. This compound alters the bioluminescence color of RLuc by substituting the sulfur and oxygen heteroatoms in the methylene bridge. Coelenteramine 400a enhances signal resolution, proving useful in bioluminescence resonance energy transfer (BRET) research [3].

Luminescent reaction
I. Solution preparation
1. Mother solution preparation: Use ethanol to prepare Coelenteramine 400a into a mother solution of appropriate concentration (1-10mM). The concentration of the mother solution can be adjusted according to the specific experimental conditions.
2. Working solution preparation: Dilute it with water/PBS/DMEM to the required working solution when using.
II. Operation steps
1. Mix 10 μL of Coelenteramine 400a solution with a concentration of 1.0-5.0 mM and 10 μL of Renilla luciferase (RLuc8.6-53) solution with a concentration of 2.01 mg/mL with 200 μL buffer.
2. Use a multi-channel spectrometer equipped with a liquid nitrogen-cooled CCD detector to record the bioluminescence spectrum of the mixed solution.
Note: The absolute spectral sensitivity of the spectrometer needs to be calibrated in advance with a 500 W spectral irradiance standard lamp.
3. A custom-made photometer equipped with a photomultiplier tube (PMT, H11890-01, Hamamatsu Photonics, Japan) was used to measure the relevant parameters of the bioluminescent reaction.
Note: The absolute response of the photometer needs to be calibrated with an integrating sphere spectrometer and is determined by plotting a linear graph between the relative counts measured by the photometer and the absolute values ​​measured by the integrating sphere.
4. In a test tube, 5-15 μL of a 10 nM (50-150 fmol in total) Coelenteramine 400a solution was 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) was injected to start the reaction. The reaction was monitored until the luminescent reaction was complete, and the quantum yield of the bioluminescent reaction was calculated from the total number of photons obtained and the number of luciferin molecules.
5. The Michaelis constant (Km) of the substrate coelenteramine 400a was calculated using the Lineweaver-Burk plot. The catalytic constant (kcat) was 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. [4]