Abstracts

Luciferases are the enzymes which catalyze the oxidation reactions of luciferins responsible for bioluminescence in different organisms. Beetle luciferases have been widely used for detection of ATP, enzymatic assays, microbiological contamination, as reporter genes for bioimaging and biosensors. More recently their pH-sensitivity has been harnessed for ratiometric indication of intracellular pH and heavy metals. Our research group has cloned the largest variety of beetle luciferases, some of them already having bioanalytical applications. However, among our luciferase gene bank, there are still several luciferases with potential analytical applicability that require characterization. The luciferase from the Atlantic rain forest Cratomorphus distinctus firefly (CrtLuc) was already cloned (VIVIANI et al., 2004), however, its spectral and kinetic properties were not characterized in detail. In this work we characterized the kinetic and spectral properties of C. distinctus luciferase, explored its suitability as a luminescent sensor for pH and heavy metals and compared with other firefly luciferases cloned by our group (Macrolampis sp, Amydetes vivianii, Aspisoma lineatum and Bicellonycha lividipennis). This luciferase was expressed in E. coli BL21-DE3 and purified by Nickel affinity chromatography. The kinetic parameters (specific activity, k_cat, K_M for substrates and optimum pH) were determined through bioluminescence intensities measurements using an AB-2200 luminometer (ATTO) and their bioluminescence spectra and sensitivity to pH and metals were measured using an AB-1850 spectroluminometer (ATTO). The specific activity was high when compared to other firefly luciferases (5.5.10¹¹ cps.mg⁻¹), with k_cat = 7.0. 10 ⁻⁵ cps. The optimum pH was ~8.2, K_M(LH₂) = 16 µM and K_M(ATP)=24 µM. The low K_M and high k_cat values ​​indicate that this luciferase has high catalytic efficiency (k_cat/K_M =4.4 c.s^-1µM^-1 for LH₂ and 2.9 c.s^-1µM^-1 for ATP ). The bioluminescence spectrum of this luciferase has an emission peak at 564 nm at pH 8.0, and 611 nm at pH 6.0, showing that this luciferase is highly sensitive to pH. Noteworthy, mercury was the only metal which affected the bioluminescence spectrum, causing a shift of 50 nm at the concentration of 2 mM, demonstrating an unusual sensitivity of this luciferase to this metal when compared to other firefly luciferases. Thus, these results indicate that C. distinctus luciferase has potential applicability as a sensor color tuning luciferase for mercury detection, and as also as an indicator of intracellular pH.