IDENTIFICATION OF A LUMAZINE PROTEIN FROM PHOTOBACTERIUM LEIOGNATHI BY COHERENT ANTI-STOKES RAMAN SPECTROSCOPY

The vibrational fingerprint in the frequency region 1700-1100 cm^-1 has been obtained for a fluorescent protein purified from the bioluminescent bacterium Photobacterium leiognathi. using the technique of resonance coherent anti-Stokes Raman scattering. The frequencies of the 4 prominent modes are the same as for lumazine protein purified from Photobacterium phosphoreum , and for an authentic sample of 6,7-dimethyl-8-ribityllumazine. The fluorophore in the new protein, therefore, must have the same chemical structure. The absence of frequency shifts between the free and bound ligand, in contrast to observations of fiavoproteins, implies that the lumazine ring undergoes no changes in hydrogen bonding between the free and protein-bound ligand in its ground electronic state. However, changes in relative intensities of two modes are seen, arising from differences in interactions in the excited state, and this is also reflected by differences in fluorescence properties.