Bioluminescence decay kinetics in the reaction of bacterial luciferase with different aldehydes

Abstract

At 22 degrees C the bioluminescence decay kinetics in the in vitro reaction catalysed by Vibrio harveyi luciferase in the presence of different aldehydes--nonanal, decanal, tridecanal and tetradecanal did not follow the simple exponential pattern and could be fitted to a two-exponential process. One more principal distinction from the first-order kinetics is the dependence of the parameters on aldehyde concentration. The complex bioluminescence decay kinetics are interpreted in terms of a scheme, where bacterial luciferase is able to perform multiple turnovers using different flavin species to produce light. The initial phase of the bioluminescent reaction appears to proceed mainly with fully reduced flavin as the substrate while the final one results from the involvement of flavin semiquinone in the catalytic cycle.

Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes

“…Understanding the origin and kinetic behavior of the two phases of the response profile is essential for interpreting the luminescence signals. Luciferase-substrate complexes are believed to lead to the formation of long-lived intermediates (15,20). By looking at the biochemistry, it is reasonable to hypothesize that equation 1, as well as equation 2, would reach equilibrium in the presence of oxygen prior to the decanal addition.…”

“…If this is indeed the case, a certain proportion of the luciferase is present as a luciferase-FMNHOOH complex prior to decanal addition. The injection of decanal then allows the fast reactions leading to luminescence to proceed, as shown in equations 3 and 4 (20). Consequently, the initial luminescence intensity should be proportional to the amount of preformed luciferase complex.…”

“…Several other practical problems also arise in using decanal as an in vivo assay reagent: decanal is highly volatile, it is poorly soluble in aqueous systems, it is lipophilic (and hence tends to partition into lipid phases within cells), and it is cytotoxic at concentrations above a few hundred micromolar. Shorter-chain aldehydes and other proposed substitutes have been studied (15,20,31,37), but decanal appears to be the optimal, or nearly optimal, substrate from the point of view of the biochemistry of the luciferase, at least over a narrow but well characterized concentration range. Most workers will therefore continue to show a preference for using decanal, but it is critical to specify an appropriate concentration and to carefully design the assay so that the concentration remains well defined during data acquisition.…”