STRUCTURAL CHARACTERISTICS OF FLUOROPHORES THAT FORM INTRAMOLECULAR H-TYPE DIMERS IN A PROTEASE SUBSTRATE

Recently, we designed and synthesized a new class of profluorescent pr otease substrates whose spectral properties fit the exciton model: mor e specifically, spectra of these polypeptide which were doubly labeled with rhodamines showed blue shifted absorption peaks and fluorescence quenching, both indicators of H-dimer formation. In the work describe d here NorFES, an undecapeptide which is cleaved by the serine proteas e elastase, was homodoubly labeled on opposite sides of its cleavage s ite with six fluorophores in order to identify structural elements of dyes which influence intramolecular H-type dimer formation, Absorption and fluorescence spectra of these six substrates obtained before and after enzymatic cleavage indicate that the exciton band is strongest i n the peptide doubly labeled with tetramethylrhodamine, followed by rh odamine-X. and then (diethylamino)coumarin. In contrast, spectra of No rFES homodoubly labeled with fluorescein, hydroxycoumarin, or pyrene d o not exhibit exciton bands, These data suggest that factors significa nt in H-type dimerization are as follows (in decreasing order): deloca lized charge, symmetry. and magnitude of the lowest energy electronic transition dipole. Surprisingly, in the group of fluorophores in this study, no evidence for hydrophobic interactions as an important influe nce was observed.