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Issue 9, 2008
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Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate

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Abstract

The inhibition mechanisms of the firefly luciferase (Luc) by the two major products of the reactions catalysed by Luc, oxyluciferin and dehydroluciferyl-adenylate (L-AMP), were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 μM oxyluciferin; 0.0025 to 1.25 μM L-AMP) has been measured in 50 mM Hepes buffer (pH = 7.5), 10 nM Luc, 250 μM ATP and D-Luciferin (from 3.75 up to 120 μM). Nonlinear regression analysis with the appropriate kinetic models (Henri–Michaelis–Menten and William–Morrison equations) reveals that oxyluciferin is a competitive inhibitor of luciferase (Ki = 0.50 ± 0.03 μM) while L-AMP act as a tight-binding competitive inhibitor (Ki = 3.8 ± 0.7 nM). The Km values obtained both for oxyluciferin and L-AMP were 14.7 ± 0.7 and 14.9 ± 0.2 μM, respectively. L-AMP is a stronger inhibitor of Luc than oxyluciferin and the major responsible for the characteristic flash profile of in vitro Luc bioluminescence.

Graphical abstract: Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate

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Publication details

The article was received on 11 Jun 2008, accepted on 21 Jul 2008 and first published on 01 Aug 2008


Article type: Paper
DOI: 10.1039/B809935A
Citation: Photochem. Photobiol. Sci., 2008,7, 1085-1090
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    Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate

    C. Ribeiro and J. C. G. Esteves da Silva, Photochem. Photobiol. Sci., 2008, 7, 1085
    DOI: 10.1039/B809935A

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