The intrinsic fluorescence of apo-obelin and apo-aequorin and use of its quenching to characterize coelenterazine binding

Eremeeva, Elena V.; Markova, Svetlana V.; Westphal, Adrie H.; Visser, Antonie J.W.G.; Berkel, Willem J.H. van; Vysotski, Eugene S.

doi:10.1016/j.febslet.2009.04.043

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Volume 583, Issue 12 , Pages 1939-1944, 18 June 2009

The intrinsic fluorescence of apo-obelin and apo-aequorin and use of its quenching to characterize coelenterazine binding

Edited by Richard Cogdell

Elena V. Eremeeva
- Photobiology Laboratory, Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia
- Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands
Svetlana V. Markova
- Photobiology Laboratory, Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia
Adrie H. Westphal
- Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands
Antonie J.W.G. Visser
- Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands
Willem J.H. van Berkel
- Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands
Eugene S. Vysotski
- Photobiology Laboratory, Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia
- Corresponding author. Fax: +7 391 2433400.

Received 24 March 2009; received in revised form 23 April 2009; accepted 28 April 2009. published online 06 May 2009.

Abstract

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±0.12μM) and apo-obelin (0.2±0.04μM). 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.