Coelenterazine is a naturally occurring substrate, or luciferin, for a diverse collection of luciferases. In the presence of molecular oxygen, the luciferase oxidizes coelenterazine, generating a high-energy intermediate and emitting blue light in the process (1). Unlike beetle luciferin-luciferase systems, coelenterazine luciferases do not require ATP and are often more convenient for in vivo bioluminescence experiments (2). The best-characterized coelenterazine luciferase comes from the sea pansy Renilla.

Coelenterazine is also the chromatophore cofactor of another family of marine photoproteins, exemplified by aequorin from the Aequorea jellyfish. In a variation of the luciferin-luciferase reaction, aequorin is “preloaded” with a stably bound, reactive form of coelenterazine and the oxidation reaction proceeds upon binding of Ca2+ to the aequorin protein. This response to calcium is essentially oxygen-independent and the amount of blue light emitted by aequorin is proportional to the enzyme and Ca2+ concentrations (1).

Coelenterazine is hydrophobic and can easily cross cell membranes, making it amenable to use in whole-cell experiments. In addition to native coelenterazine, several analogs of coelenterazine with altered emission properties or chemical stabilities are commercially available. As a substrate for Renilla luciferase (Rluc), native coelenterazine emits light at ~460nm. In comparison, coelenterazine 400a, a commonly used analog often referred to as DeepBlueC, emits light at ~395nm but decays rapidly in aqueous solution(3)(4).

The coelenterazine-aequorin bioluminescence reaction is sensitive to even slight changes in Ca2+ concentrations and has been used extensively as a biosensor for intracellular calcium(5). Unlike calcium-sensitive dyes, recombinant aequorin can be targeted to specific cellular compartments, allowing for subcellular resolution of calcium measurements. Wild-type aequorin can be used to measure calcium concentrations that fall between 0.5µM-10µM and mutations that lower the protein’s affinity for calcium can expand this range up to 100µM(5).

Bioluminescence resonance energy transfer (BRET) is a recently developed experimental technique that also exploits coelenterazine-based bioluminescence (6) (7). Like fluorescence resonance energy transfer (FRET), BRET involves the transfer of energy from a light-emitting donor to a fluorescent acceptor, resulting in a shift in the detectable light spectrum(8). This energy transfer is only possible over distances less than 10nm, making it an effective gauge of protein interactions when the donor and acceptor proteins are fused to suitable targets. Unlike FRET, BRET uses an enzymatic source of luminescence not external optical excitation, so it avoids several common FRET complications like photobleaching, autofluorescence, and tissue damage from laser exposure(8). BRET has been used successfully in bacteria, yeast, plants, and mammalian cells and has proven especially valuable for studying real time interactions, the mechanisms of G- protein coupled receptors, and high variation of the originally published BRET technique, uses Rluc and GFP, as the donor and acceptor respectively, and coelenterazine 400a as the substrate.

The BRET technique can be further optimized to suit a range of experimental systems, instrumentation, and conditions (6). The use of different coelenterazine substrates influences the quantum yield of the donor reaction, the overlap of donor and acceptor spectra, or the rate of signal decay. Also, mutants of Rluc exist that alter its emission spectrum or increase its stability in cell culture conditions (2).

1. Wilson T, Hastings JW. BIOLUMINESCENCE. Annu. Rev. Cell. Dev. Biol. 1998;14(1):197-230.

2. Loening AM, Fenn TD, Wu AM, Gambhir SS. Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Engineering, Design and Selection 2006 Sep;19(9):391-400.

3. Pfleger KDG, Seeber RM, Eidne KA. Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions. Nat Protoc 2006;1(1):337-345.

4. DeepBlueC is a trademark of the Packard BioScience Company.

5. Brini M. Calcium-sensitive photoproteins. Methods 2008 Nov;46(3):160-166.

6. Bacart J, Corbel C, Jockers R, Bach S, Couturier C. The BRET technology and its application to screening assays. Biotechnology Journal 2008;3(3):311-324.

7. Pfleger KDG, Eidne KA. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Meth 2006 Mar;3(3):165-174.

8. Xu Y, Piston DW, Johnson CH. A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins. Proceedings of the National Academy of Sciences of the United States of America 1999 Jan;96(1):151-156.

9. Kocan M, Pfleger KD. Detection of GPCR/β-Arrestin Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer Technology [Internet]. In: G Protein-Coupled Receptors in Drug Discovery. 2009 p. 305-317.[cited 2010 Feb 2 ] Available from:

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