Temperature effect on the bioluminescence spectra of firefly luciferases: potential applicability for ratiometric biosensing of temperature and pH
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.