Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum

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Abstract
The properties of lumazine proteins purified from the marine bioluminescent bacteria P. phosphoreum, a psychrophile, and P. leiognathi, a relatively thermophilic species, are compared. An accurate 1:1 stoichiometry of binding of the ligand 6,7-dimethyl-8-ribityllumazine to each lumazine protein is established by back-titration of the apoprotein with the same authentic ligand, using both fluorescence and absorption measurements. Neither protein contains metal cofactors, organic P, or carbohydrate. Both proteins are anionic and hydrophilic. They each contain a single Trp residue and have blocked amino terminals but otherwise differ in amino acid composition and other properties (P. phosphoreum and P. leiognathi, respectively): Met (internal), 1, 2; Cys, 2, 1; Arg, 4, 7; isoelectric point, 4.78 and 4.83, 4.38 and 4.45; MW 19,750, 21,300. In the P. phosphoreum protein both Cys residues are accessible, but in the P. leiognathi protein the single Cys is buried. Modification of this buried Cys and at least 1 Cys in the P. phosphoreum protein prevents binding of the ligand. The UV and visible absorption spectra of both lumazine proteins denatured in 6 M guanidine hydrochloride can be accurately modeled by using the number of equivalents of the lumazine derivative and blocked aromatic amino acid model compounds determined by chemical and spectrophotometric analyses for Trp, Tyr and Phe.

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