Upon binding the substrate, the enzyme catalyzes an oxygenation, producing a very short-lived hydroperoxide that cyclizes into a dioxetanone structure, which collapses, releasing a CO2 molecule. The spontaneous breakdown of the dioxetanone releases the energy (about 50 kcal/mole) that is necessary to generate the excited state of the coelenteramide product, which is the singlet form of the monoanion. In vivo the product undergoes the process of nonradiative energy transfer to an accessory protein, a green fluorescent protein (GFP), which results in green bioluminescence. In vitro, in the absence of GFP, the product emits blue light.
Background References
1. Shen L et al. Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene. Antimicrob Agents Chemother 60:5492-503 (2016).
2. Loening AM et al. Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis. J Mol Biol. 374(4):1017-28 (2007).
Western blot analysis of Renilla Luciferase on luciferase recombinant protein. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1602-45, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.
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