Photochemical & Photobiological Sciences PAPER Cite this: Photochem. Photobiol. Sci., 2019, 18, 2682 Received 6th June 2019, Accepted 22nd August 2019 DOI: 10.1039/c9pp00257j rsc.li/pps Temperature effect on the bioluminescence spectra of firefly luciferases: potential applicability for ratiometric biosensing of temperature and pH Gabriela Oliveira a,b and Vadim R. Viviani * a,b Bioluminescence spectra of firefly luciferases are affected by pH, heavy metals and high temperatures. Previously, we compared the effect of pH and heavy metals on the bioluminescence spectra of different firefly luciferases and showed that such spectral sensitivity can be harnessed to ratiometrically estimate the pH inside cells and metal concentration. Here, we compared the effect of temperature on the spectral sensitivity of four firefly luciferases (Amydetes vivianii: 539 nm; Cratomorphus distinctus: 548 nm; Photinus pyralis: 558 nm and Macrolampis sp2: 594 nm) and investigated whether a ratiometric curve could be used to estimate temperature. The ratio of intensities of bioluminescence at two wavelengths (green and red) at different temperatures (5–35 °C) was determined. The results confirm that, in the case of pH-sensitive luciferases, the more blue-shifted the bioluminescence spectrum, the more thermostable the enzyme and the less sensitive the emission spectrum to temperature. An almost linear relationship between temperature and the ratio of bioluminescence intensities in the green and red region of the spectrum was found for the four luciferases: the more blue-shifted and less sensitive luciferases exhibit a smaller slope and the more red-shifted luciferases exhibit a steeper slope in the following order: Amy < Crt < Ppy < Mac. This relationship offers the possibility of using firefly luciferases as ratiometric indicators of temperature and may allow the compensation of the effect of temperature in the ratiometric analysis of intracellular pH and heavy metal concentration for each enzyme. Introduction Firefly luciferases catalyze the ATP dependent oxidation of a benzothiazolic luciferin emitting yellow-green light at physio- logical pH. 1,2 However, at acidic pH, in the presence of heavy metals and at high temperatures they produce red light with lower efficiency. 3,4 Click beetle and railroad worm luciferases emit a wide range of colors from green to red, but their spectra are insensitive to pH, heavy metals and temperature. 2 Bioluminescence colors are determined by the microenvi- ronment of the active site of the luciferase 1,2 since the luciferin and the bioluminescence reaction are the same for the three bioluminescent families of Coleoptera. In general, non-specific polarity effects, specific-acid–base and electrostatic inter- actions and conformational changes have been claimed to explain how luciferase active sites determine bioluminescence colors. 2,5–7 Several studies have been conducted to understand the influence of luciferase structures on bioluminescence colors and pH-sensitivity. 6,8–11 Very recently, the pH-sensing and metal binding site of firefly luciferases was identified. 12 Such a binding site involves the electrostatic couples H310/ E354 and E311/R337 which bind metals and are affected by pH. 13 Firefly luciferases have been widely applied for bioanalytical purposes including the analysis of the microbiological con- tamination of biological fluids, evaluation of cell viability, and enzymatic assays involving the generation or degradation of ATP. 5,7,8 The number of applications has increased with the use of luciferase reporter genes in the analysis of the transcrip- tional activity of promoters in different cells, 14–18 including mammalian cells, and as multiple reporter systems that use distinct luciferases emitting different bioluminescence colors for simultaneous analysis of multiple cellular events. 19,20 More recently, pH-sensitivity and firefly luciferases were shown to be useful to ratiometrically indicate intracellular pH in live bacteria and mammalian cells, 21–23 and then to esti- mate heavy metal concentrations. Although the effect of pH and heavy metals has been studied in more detail for some a Laboratory of Biochemistry and Biotechnology of Bioluminescence, Graduate Program of Biotechnology and Environmental Monitoring, Department of Chemistry, Physics and Mathematics, Federal University of São Carlos (UFSCar), Campus of Sorocaba, Sorocaba, SP, Brazil. E-mail: viviani@ufscar.br; Fax: +55-1532295983; Tel: +55-1532295983 b Department of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil 2682 | Photochem. Photobiol. Sci. , 2019, 18, 2682–2687 This journal is © The Royal Society of Chemistry and Owner Societies 2019