Separation and structure of the prosthetic group of the blue fluorescence protein from the bioluminescent bacterium Photobacterium phosphoreum

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Abstract
The highly fluorescent prosthetic group of the blue fluorescence protein purified from the bioluminescent bacterium P. phosphoreum was dissociated and separated from its apoprotein by affinity chromatography on Cibacron Blue-Sepharose. It was identified as 6,7-dimethyl-8-(1''-D-ribityl)lumazine by several methods of characterization, all of which gave results identical to those for an authentic sample. In neutral solution, absorption maxima are at 407, 275 (shoulder) and 256 nm, with a single fluorescence maximum at 491 nm. The PMR spectrum exhibits a singlet at 2.66 ppm corresponding to the methyl substituted at the 6 position of lumazine and a multiplet centered at 3.85 ppm corresponding to the C-2''-5'' protons of the ribityl group. A Raman spectrum was obtained by the technique of coherent anti-Stokes Raman scattering and the R. values by paper chromatography were determined in 4 solvent systems. The isolated compound was readily transformed into riboflavin by riboflavin synthetase. Fifty grams (dry weight) of P. phosphoreum contains at least 20 mg of this lumazine derivative, an amount comparable to that found in other microorganisms classified as riboflavin overproducers. The overproduction of this lumazine in this case apparently has to do with its function in the generation of bioluminescence.