Fluorescence anisotropy decay study of self-association of bacterial luciferase intermediates

The fluorescence dynamics parameters of the fluorescent transient flavin-luciferase species from the types Vibrio fischeri and Photobacterium leiognathi are presented. The fluorescence anisotropy decay is a single exponential function for both types. The correlation time is 70 ns for the P. leiognathi fluorescent transient intermediate (2°C, aqueous buffer, pH 7.0), consistent with the rotational correlation time of the luciferase macromolecule (77 kD) to which the flavin fluorophore is rigidly attached. In contrast, for the V. fischeri species the observed correlation time for the anisotropy decay function is 133 ns. This suggests that protein self-association occurs in the V. fischeri case and this is confirmed by filtration, where the fluorescent transient from V. fischeri does not pass through a 100,000 molecular weight cutoff membrane, whereas the P. leiognathi species does. The filtration method also demonstrates self-association in the luciferase peroxyflavin and photoflavin from V. fischeri. A monomer-dimer equilibrium also explains the previously reported high correlation times for the V. harveyi luciferase-flavin species. It is proposed that the self-association competes with the lumazine protein interaction in the bioluminescence reaction. © 1991 Plenum Publishing Corporation.