Inhibition sensitivity of <i>in vitro</i> firefly bioluminescence quantum yields to Zn²⁺ and Cd²⁺ concentrations in aqueous solutions

To elucidate the inhibition effects of Zn²⁺ and Cd²⁺ on the luciferin-luciferase reaction, we performed quantitative measurements of quantum yields and spectral shapes for in vitro firefly bioluminescence in aqueous solutions containing ZnSO₄, ZnCl₂, CdSO₄, and CdCl₂ at different concentrations. Particular care was taken toward the equilibrium between metal ions and enzyme proteins, anion difference, solubility, and uncertainty evaluation. The bioluminescence quantum yields decreased almost linearly to the concentration of Zn²⁺ and Cd²⁺ below 0.25 mM. No obvious difference was found between the chloride and sulfate anion solutions. We defined inhibition sensitivity as the decrease in relative quantum yield versus the concentration of metal ions, and they were determined to be 1.48 ± 0.13 and 1.13 ± 0.16/mM for Zn²⁺ and Cd²⁺, respectively. We estimated the detection limit of inhibition effects as the concentration of metal ions that decrease relative quantum yields by 10%, which were 0.07 mM (4 ppm) and 0.09 mM (10 ppm) for Zn²⁺ and Cd²⁺, respectively. The shape of the bioluminescence spectra changed sensitively with the increase in Zn²⁺ concentrations. The bioluminescence peak energy for 0.10-mM Zn²⁺ was ~2.2 eV, while that for 0.25-mM Zn²⁺ was ~2.0 eV. The shape of the spectra changed less sensitively with the increase in Cd²⁺concentrations, and the peak energy was at ~2.2 eV for Cd²⁺ concentrations of 0.10 and 0.25 mM.