Spontaneous luminescence color change in the firefly luciferase assay system

The temporal effects of luciferase reaction luminescence have only been discussed in the context of light intensity (flash vs. glow). However, alterations in the color of the light emitted over the course of the luciferase reaction have not been reported. Here, we show a temporal change in the light color emitted during the reaction catalyzed by unmodified firefly luciferase when concentrations of one of the substrates, adenosine triphosphate (ATP), are gradually increased. The temporal color change from green to red occurs within the first few minutes of the luciferase reaction when an ATP-containing solution is either added or synthesized in situ with the aid of an autocatalytic reaction occurring simultaneously. This color change is not accompanied by pH changes. An analysis of the red and green channels demonstrates dissimilar kinetics, suggesting the co-existence of two or more temporally shifted luminescence pathways. The implications of these findings might improve dual-color biosensing/imaging protocols and influence the engineering of biophotonic systems.