Inouye Satoshi, Miura-Sahara Yuiko, Iimori Rie, Sakata Yuki, Hazama Yuki, Yoshida Suguru, Nakamura Mitsuhiro, Hosoya Takamitsu
Yokohama Research Center, JNC Corporation, 5-1 Okawa, Kanazawa-ku, Yokohama, 236-8605, Japan.
Yokohama Research Center, JNC Corporation, 5-1 Okawa, Kanazawa-ku, Yokohama, 236-8605, Japan.
Biochem Biophys Res Commun. 2020 May 28;526(2):404-409. doi: 10.1016/j.bbrc.2020.03.085. Epub 2020 Mar 27.
Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λ = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-HO system, but not in sodium hypochlorite.
磷光蛋白被归类为一种光蛋白，由脱辅基磷光蛋白（磷光蛋白的一种脱辅基蛋白）和一种未知的辅基作为发光源组成。磷光蛋白的发光反应由活性氧触发。重组脱辅基磷光蛋白最近被表达为谷胱甘肽S-转移酶的融合蛋白（GST-脱辅基磷光蛋白），并从大肠杆菌细胞中纯化出来。通过将非荧光的脱氢腔肠素（dCTZ，CTZ的脱氢形式）与GST-脱辅基磷光蛋白一起孵育，GST-脱辅基磷光蛋白和dCTZ的复合物（GST-脱辅基磷光蛋白/dCTZ复合物）立即形成，并显示出亮黄色荧光（λ = 539 nm，在430 nm激发）。出乎意料的是，GST-脱辅基磷光蛋白/dCTZ复合物的荧光发色团被鉴定为非荧光的dCTZ。GST-脱辅基磷光蛋白/dCTZ复合物的发光强度在过氧化氢酶-HO系统中增加，但在次氯酸钠中没有增加。
