Selective modification of Trp19 in β-lactoglobulin by a new diazo fluorescence probe

To obtain the local information on the tryptophan domain in a protein, the design and synthesis of a new fluorescent probe, 1,7-bis(4-hydroxy-3-methoxyphenyl)-4-diazo-1,6-heptadiene-3,5-dione, is reported for the Selective modification of tryptophan residues. The probe comprises a curcumin fluorophore and a diazo labeling group, whose spectroscopic properties are characterized. The diazo group may be catalytically degraded by transition metal complexes such as Rh-2(OAc)(4), generating an active rhodium carbenoid intermediate, which can react selectively with tryptophan residues. By the use of the carbene's intermolecular reactions, the tryptophan residue (Trp19) of beta-lactoglobulin may be modified with the diazo curcumin probe. Furthermore, slight secondary but larger tertiary structural changes are detected after Trp19 is modified, and the Trp19 modification produces a great effect on the binding of 8-anilino-1-naphthalenesulfonic acid and retinol. These results indicate that the Trp19 residue plays an essential role in the structure and stability of beta-lactoglobulin, and the specific modification of this residue may have a potential use in further elucidating the relationship between the structure and function of the protein.