Lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. A fluorescence study of the protein-ligand equilibrium

Abstract

The changes of fluorescence spectral distribution, polarization and lifetime of the lumazine protein from P. phosphoreum can be interpreted in terms of an equilibrium between the protein and its dissociated prosthetic group 6,7-dimethyl-8-(1''-D-ribityl)lumazine. The equilibrium is rapidly attained, 1:1, and Kd is 5 .times. 10-8 M (4.degree. C, pH 7, 67 mM phosphate). A change in solution conditions like an increase in temperature or dilution or a decrease in pH or ionic strength favors dissociation of the ligand from the protein. The dissociation was confirmed by separating the free ligand by ultrafiltration, and the apoprotein was reconstituted with the authentic lumazine derivative. A van''t Hoff analysis of the Kd allows evaluation of the thermodynamic parameters: .DELTA.H.degree. [standard entalpy change] = 28 kcal/mol and .DELTA.S.degree. [standard entropy change] = 67 eu [entropy units]. By analogy to published results on the binding of FMN to apoflavodoxin, the contribution of hydrophobic interaction and of H bonding to the binding enthalpy can be estimated. The decay of the emission anisotropy of lumazine protein following a 0.5-ns laser pulse excitation can be fitted with a single correlation time characteristic of a 30,000-dalton spherical protein.