The luminescence activity of BFP (Ca2+-bound apoaequorin–coelenteramide complex) is about 10 times higher than that
prothrombin 474-477 of the Ca2+-bound apoaequorin [9]. Thus, BFP is a bifunctional protein possessing blue fluorescence and the catalytic activity of luminescence. Further, BFP is a heat-resistant protein [9] and is converted to a greenish fluorescent protein (gFP) with the maximum peak around 470 nm by treatment of a chelating reagent such as EDTA. It is interesting that gFP is a non-covalent complex of apoaequorin with coelenteramide and can be regenerated to aequorin by incubation of coelenterazine in the absence of reducing reagents at 25 °C [9]. By incubation of BFP or gFP with various coelenterazine analogues in the presence of EDTA and dithiothreitol (DTT), semi-synthetic aequorins could be also prepared [16]. Furthermore, the luminescence activity of BFP with coelenterazine and its analogues was stimulated by the addition of imidazole at concentrations of 30–300 mM [17]. However, the catalytic region or residue(s) for
oxidation of coelenterazine with molecular oxygen in aequorin and BFP were not solved. To study the structure–function relationships, it is necessary to establish the conventional method for preparing BFP and gFP. In this report, we established new chemical synthetic route of coelenteramide and reconstituted BFP (synthetic BFP: syn-BFP) and gFP (synthetic gFP: syn-gFP) efficiently from highly purified recombinant apoaequorin and coelenteramide.
