Inouye Satoshi, Nakamura Mitsuhiro, Hosoya Takamitsu
Yokohama Research Center, JNC Co. 5-1, Okawa, Yokohama, 236-8605, Japan.
Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima, 770-8506, Japan.
Photochem Photobiol. 2022 Sep;98(5):1068-1076. doi: 10.1111/php.13590. Epub 2022 Jan 12.
Aequorin consists of apoprotein (apoAequorin) and (S)-2-peroxycoelenterazine (CTZ-OOH) and is considered to be a transient-state complex of an enzyme (apoAequorin) and a substrate (coelenterazine and molecular oxygen) in the enzymatic reaction. The degradation process of CTZ-OOH in aequorin was characterized under various conditions of protein denaturation. By acid treatment, the major product from CTZ-OOH was coelenteramine (CTM), but not coelenteramide (CTMD), and no significant luminescence was observed. The counterparts of CTM from CTZ-OOH were identified as 4-hydroxyphenylpyruvic acid (4HPPA) and 4-hydroxyphenylacetic acid (4HPAA) by liquid chromatography/electrospray ionization-time-of-flight mass spectrometry (LC/ESI-TOF-MS). In the luminescence reaction of aequorin with Ca , similar amounts of 4HPPA and 4HPAA were detected, indicating that CTM is formed by two pathways from CTZ-OOH through dioxetanone anion and not by hydrolysis from CTMD.
水母发光蛋白由脱辅基蛋白（脱辅基水母发光蛋白）和（S）-2-过氧腔肠素（CTZ-OOH）组成，被认为是酶促反应中一种酶（脱辅基水母发光蛋白）和一种底物（腔肠素和分子氧）的瞬态复合物。在蛋白质变性的各种条件下，对水母发光蛋白中CTZ-OOH的降解过程进行了表征。通过酸处理，CTZ-OOH的主要产物是腔肠胺（CTM），而不是腔肠酰胺（CTMD），并且未观察到明显的发光。通过液相色谱/电喷雾电离-飞行时间质谱（LC/ESI-TOF-MS）将CTZ-OOH产生的CTM对应物鉴定为4-羟基苯丙酮酸（4HPPA）和4-羟基苯乙酸（4HPAA）。在水母发光蛋白与Ca的发光反应中，检测到了相似量的4HPPA和4HPAA，这表明CTM是由CTZ-OOH通过二氧杂环丁烷阴离子经两条途径形成的，而不是由CTMD水解形成的。
