Alteration in the Sensitivity of Firefly Bioluminescence to pH Driven by Molecular Recruitment to Coacervate Droplets

Firefly bioluminescence is widely used as a noninvasive optical imaging probe. However, the emitted light is highly susceptible to environmental factors, particularly pH, and thus, there exists a risk of misinterpretation of results from bioluminescence imaging studies depending on the local cellular environment. In this study, we investigated how the recruitment of an enzymatic reaction system into biomolecular condensates, such as membrane-less organellesone of the regulatory systems for intracellular biochemical reactionsinfluences the pH sensitivity of a bioluminescence reaction. We selected coacervates composed of homopeptides (polyarginine or polylysine) and adenosine triphosphate (ATP) to be artificial models of membrane-less organelles. Both firefly luciferase and its substrate d-luciferin were concentrated within the coacervates across a pH range of 5.1-10.0. Interestingly, in the case of the polyarginine coacervate, the recruitment of the enzymatic reaction system not only enhanced the bioluminescence intensity over a wide pH range but also markedly shifted the pH dependence of the bioluminescence color. A kinetic analysis, inhibitor assays, and quantum yield measurements revealed that the enhancement of the bioluminescence intensities resulted from a reduction in the inhibitory effects of reaction products as well as changes in the bioluminescence quantum yield. These findings provide critical insights for interpreting bioluminescence imaging results in the presence of intracellular membrane-less organelles and offer a potential avenue for developing novel biosensing tools that leverage these condensates.