2. Academic Validation
  3. Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca2+ -Binding Photoprotein Aequorin

Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca2+ -Binding Photoprotein Aequorin

  • Photochem Photobiol. 2022 Sep;98(5):1068-1076. doi: 10.1111/php.13590.
Satoshi Inouye 1,  Mitsuhiro Nakamura 2,  Takamitsu Hosoya 3
Affiliations

Affiliations

  • 1 Yokohama Research Center, JNC Co. 5-1, Okawa, Yokohama, 236-8605, Japan.
  • 2 Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima, 770-8506, Japan.
  • 3 Laboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 kanda-Surugadai, Chiyoda-ku, Tokyo, Japan.
PMID: 34971002 DOI: 10.1111/php.13590
Abstract

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 CA2+ , 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.

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