The intrinsic fluorescence of apo-obelin and apo-aequorin and use of its quenching to characterize coelenterazine binding.
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 microM) and apo-obelin (0.2+/-0.04 microM). 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.
Photobiology Laboratory, Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russia.
Elena V
EV
Eremeeva
Svetlana V
SV
Markova
Adrie H
AH
Westphal
Antonie J W G
AJ
Visser
Willem J H
WJ
van Berkel
Eugene S
ES
Vysotski
D000331:Aequorin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D037402:Hydrozoa / Q000235:genetics / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011719:Pyrazines / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D001059:Apoproteins; D007093:Imidazoles; D008164:Luminescent Proteins; D011719:Pyrazines; D011994:Recombinant Proteins; C060014:apoaequorin; C448311:apoobelin; C017144:coelenterazine; D000331:Aequorin; D014364:Tryptophan