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The intrinsic fluorescence of apo‐obelin and apo‐aequorin and use of its quenching to characterize coelenterazine binding
Author(s) -
Eremeeva Elena V.,
Markova Svetlana V.,
Westphal Adrie H.,
Visser Antonie J.W.G.,
van Berkel Willem J.H.,
Vysotski Eugene S.
Publication year - 2009
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2009.04.043
Subject(s) - photoprotein , aequorin , chemistry , fluorescence , quenching (fluorescence) , photochemistry , bioluminescence , biophysics , biochemistry , biology , physics , quantum mechanics , intracellular
The intrinsic fluorescence of two apo‐photoproteins has been characterized and its concentration‐dependent quenching by coelenterazine has been for the first time applied to determine the apparent dissociation constants for coelenterazine binding with apo‐aequorin (1.2 ± 0.12 μM) and apo‐obelin (0.2 ± 0.04 μM). Stopped‐flow measurements of fluorescence quenching showed that coelenterazine binding is a millisecond‐scale process, in contrast to the formation of an active photoprotein complex taking several hours. This finding evidently shows that the rate‐limiting step of active photoprotein formation is the conversion of coelenterazine into its 2‐hydroperoxy derivative.