71 オワンクラゲの発光蛋白質エクオリンの発光種の構造決定(ポスター発表の部)

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Aequorin (AQ) is a calcium-binding protein from the jellyfish, Aequoria victoria, which emits light (460nm) by an intramolecular reaction on binding calcium ions. AQ is composed of coelenterazine and O_2 bound to apoaequorin (apoAQ). During light cmission, coelenterazinc is oxydized to coelenteramide (1) and a blue fluorescent protein (BFP) is formed consisting of coelenteramide noncovalentry bound to apoAQ. While the singlet excited state of coelenteramide is known to be the emitter in the reaction, there has been uncertainty regarding the structure of the emitter. We here report, based on studies of coelenterazine derivatives with recombinant apoAQ, that the emitting species is a phenolate mono anion of coelenteramide. AQ was prepared by the incubating recominant apoAQ with coelenterazine, dissolved oxygen and dithiothreitol, and gave a bioluminescence emission spectrum identical to the fluorescence spectrum of the spent reaction mixture containing BFP. Incubation of recombinant apoAQ with coelenteramide also produced BFP. Molecular weight of recombinant apoAQ and BFP were confirmed by electrospray mass spectrometry. Coelenteramide has three acidic protons located at the two phenolic hydroxyls and amide groups, and structures 1a and 1b has been considered the possible emitter at 460nm. To decide between the two structures, analogues 2 and 3 were synthesized. Although the fluorescence spectrum of 2 in BFP could not be observed, 3 incubated with recombinant apoAQ yielded a BFP with a fluorescence spectrum possesing a peak at 480nm. This emission maximum is similar to bioluminescence maximum of AQ suggesting that the excited state emitter in AQ could not be the amide anion but be the phenolate anion 1a. This finding indicates that excited state formation in free solution differs from that taking place within the active site of the AQ molecule.
1993-09-10

71 オワンクラゲの発光蛋白質エクオリンの発光種の構造決定(ポスター発表の部)

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