Dynamic fluorescence study of the interaction of lumazine protein with bacterial luciferases

• Published in: Biophysical chemistry 1989-03, Vol.33 (1), p.99-111
• Main Authors: Lee, John, O'Kane, Dennis J., Gibson, Bruce G.
• Format: Article
• Language: English
• Subjects: Bacterial luciferase; Bacterial Proteins - metabolism; Bioluminescence; Carrier Proteins - metabolism; Emission anisotropy decay; Fluorescence lifetime; Fluorescence Polarization - methods; Kinetics; luciferase; Luciferases - metabolism; lumazine; Lumazine protein; Luminescent Measurements; Luminescent Proteins; Mathematics; Models, Theoretical; Photobacterium - enzymology; Photobacterium - metabolism; Vibrio - enzymology; Vibrio harveyi
• ISSN: 0301-4622; 1873-4200
• DOI: 10.1016/0301-4622(89)80012-2

The equilibrium association of lumazine protein from Photobacterium phosphoreum with luciferases from either P. phosphoreum or an aldehyde-requiring dark mutant of Vibrio harveyi is measured from changes of the rotational correlation time which is derived from the decay of the lumazine ligand's fluorescence anisotropy. The rotational correlation time of lumazine protein is 23 ns (2°C, 0.25 M P i) and is increased on addition of luciferase due to the formation of a higher molecular weight complex. The V. harveyi luciferase exhibits full competence for the association and a 1 : 1 stoichiometry, with a K d in the range 40–90 μM At lower ionic strength (0.05 M P i), the K d increases but is reduced again by the addition of dodecanol or dimyristoyllecithin. In contrast, tetradecanal, a substrate for the bioluminescence reaction, exerts no influence on the association. The equilibration rate is found to be too slow and for both luciferases the K d values are too high for the interaction of the native proteins to account quantitatively for the spectral shifting of the biolumineseence by lumazine protein.