Dye induced quenching of firefly luciferase–luciferin bioluminescence

The quenching of firefly bioluminescence (BL) in presence of xanthene dyes and tetratolylporphyrin was investigated. The BL intensity was quenched with an altered decay pattern in presence of xanthene dyes and tetratolylporphyrin. The electronic absorption spectra indicate that there is no significant interaction occurring between the dyes and the BL components in the ground state. The BL quenching decay rate and fluorescence quenching studies of luciferin by the dyes suggest an energy transfer through an exciplex, involving oxyluciferin, in the excited state and the dyes, in the ground state. The bimolecular quenching rate constant (Kq) values obtained from fluorescence studies varied between 7.7 × 1012 and 19.8 × 1012 M−1 s−1. The magnitude of the bimolecular quenching rate constants confirmed the complex formation between dye and excited oxyluciferin. The exciplex subsequently undergoes a non-radiative decay to the ground state via a combination of heavy atom induced and Förster-type energy transfer. The decay rate constants in presence and in absence of dyes vary between 7.47 × 10−4 and 7.6 × 10−2 s−1. In the presence of dyes the effective decay rate constants (keff) increased while the lifetime of light emitting species decreased. The kinetic studies in presence of singlet oxygen scavengers, like β-carotene and NaN3, prove that there is no significant quenching of the firefly BL due to the formation of singlet oxygen.