화학공학소재연구정보센터
Journal of Physical Chemistry B, Vol.118, No.8, 2070-2076, 2014
Spectroscopic Study of Firefly Oxyluciferin in an Enzymatic Environment on the Basis of Stability Monitoring
To understand the influence of the enzyme microenvironment on the properties of the emitter oxyluciferin (OL) in firefly bioluminescence, we investigated the spectroscopic characteristics of OL in a complex with the enzyme luciferase formed in a consumed reaction mixture. By monitoring the in situ absorption spectra, we analyzed the enzymatic synthesis and the stability of OL in luciferase environment. The absorption spectra of OL in Photinus pyralis luciferase showed that the dominant form was neutral OL, probably the enol form, which emitted blue fluorescence (similar to 450 nm). A monoanionic OL emitting green fluorescence (similar to 560 nm) exhibited a weak pH-dependent equilibrium with the neutral enol-OL. The red-emitting form of OL was almost completely absent from the consumed reaction mixture. The peak wavelengths of the green and red emissions of the fluorescence and bioluminescence were similar, but the peak intensities, and hence the spectral shapes, differed greatly. The above characteristics were also found in the absorption and fluorescence spectra of OL in a complex with the H433Y mutant of Lucia la cruciata luciferase, which catalyzes pH-independent red bioluminescence. Optical excitation could not reproduce the excited states of bioluminescence that was generated from the chemical reaction. The probable reason is that the chemical excited states formed from a keto-like transition state after decomposition of a dioxetanone intermediate, whereas the optical excited states were generated by exciting the neutral enol-OL. Different luciferases only influenced the chemical transition state during the bioluminescence reaction; they did not influence the ground states or optical excited states after the reaction.