Bioluminescence of Firefly Luciferase in the Presence of Biologically Relevant Cations

Abstract

The bioluminescent protein firefly luciferase is a possible biological intracellular pH reporter because it has light emission dependent on the pH of its environment. For firefly luciferase to be reliable as an intracellular pH reporter, its emission should not be significantly disturbed by the presence of biologically relevant cations. To evaluate firefly luciferase’s behavior in an environment where K+, Na+, Mg2+, and Ca2+ are present, luciferase was combined with its substrate and different concentrations of these cations in a microplate and analyzed on a plate reader to determine the emission of 550±10 and 610±10 nm light, which was used to obtain a 550:610 nm ratio. It was discovered that the plate reader’s sensitivity to low levels of bioluminescence was inconsistent, meaning that more experimentation with solutions containing higher concentrations of firefly luciferase is needed to confidently conclude that biologically relevant cations do not significantly alter the bioluminescence of firefly luciferase. However, the experiments with monovalent cations produced promising results that fell within the expected range of magnitude for the plate reader and showed small fluctuations in the 550:610 nm ratio. Although the results for divalent cations were inconclusive, firefly luciferase is still relevant for further study of possible intracellular pH reporters.