Colorful Bioluminescence: Exploring ATP’s Effect On The Firefly Luciferase Reaction

Every spring, the forest near Hualien (Taiwan) gleams with a myriad of flickering lights. Curious visitors stare at these moving bright dots with amusement. The light is produced in a bioluminescent chemical reaction occurring in the bodies of some insect species, and catalyzed by an enzyme called luciferase.

For decades now, the components of this reaction have been isolated and used by scientists in their laboratories. The reaction consumes energetic molecules – adenosine triphosphate (ATP). An enzyme called luciferase uses ATP to produce an unstable intermediate – excited state oxyluciferin. The latter one rapidly decomposes emitting photons of visible light.

One of the most widely-studied luciferases is the one isolated from firefly (Photinus pyralis). It was shown in many studies that various factors (such as pH, heavy metals, conformational changes in enzyme, or replacement of certain amino acids in enzyme) influence the color (or spectrum) of the light produced in the course of the firefly’s luciferase reaction. Various research groups have put forward hypotheses on the causes of multicolor light emission by firefly luciferase. However, they have not yet reached a consensus on the universal mechanism that controls the color of light emitted by firefly luciferase.

A recent study shows that ATP itself can affect the color of light produced in the firefly luciferase reaction. In one experiment, the researchers increased ATP concentration by continuously pipetting ATP. The reaction mixture initially emitted green light. Then, the color of light changed to red. In another experiment, they combined luciferase with other enzymes that are capable of rapidly synthesizing ATP. The resulting ATP ramp triggered a gradual change of light color from green to red.

Apart from the cognitive value of this kind of research, understanding multicolor light emission by luciferase enzyme has practical implications. That is because scientists working in various fields (biochemistry, chemistry, and molecular biology) take advantage of luciferase as a reporter molecule; for example, to measure ATP concentration, to find traces of bacterial contamination in food and environmental samples, to study the efficacy of drugs, or to verify expression of certain genes of interest. An unexpected change of color of the emitted light (appearance or disappearance of certain spectral bands) could bias the acquired data and possibly lead to incorrect conclusions. That is especially important in the context of ATP fluctuations inside cells, which might affect intracellular luciferase reporters.

On the positive side, many of these assays are performed at low concentrations of ATP, for which no significant color change was observed. When analyzing samples with high concentrations of ATP, one could possibly take advantage of the color change apart from the brightness of the emitted light to provide information on the quantity of ATP. Tuning the color of bioluminescence may also find application in bioengineered systems, in which a chemical process needs to be combined with specific response (here, it would be the emission of light of different colors). However, these prospective applications remain to be explored. As the mechanism of the light color change due to ATP increase is still elusive, the researchers are also planning to conduct further work to shed light on it.

Fig. 1: Reprinted from Analytical Biochemistry, 539, Liu P.-H., Urban P.L., Spontaneous luminescence color change in the firefly luciferase assay system, 54-59, Copyright (2017), with permission from Elsevier.

These findings are described in the article entitled Spontaneous luminescence color change in the firefly luciferase assay system, recently published in the journal Analytical Biochemistry. This work was conducted by Pawel Urban from the National Tsing Hua University and Pei-Han Liu from the National Chiao Tung University.