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NorFES (DAIPN1SIPKGY, N1 = norleucine) is an undecapeptide that contains a recognition sequence and cleavage site for the serine protease elastase. When NorFES is doubly labeled with a variety of fluorophores on opposite sides of this amino acid sequence, the fluorescence is quenched due to formation of intramolecular ground-state dimers. Although the spectral characteristics of these dimers are predictable by exciton theory, influence of the peptide backbone on H-dimer formation is less well understood. Specifically, factors that modify the attractive forces between and orientation of dyes are not well-characterized. Thus, by varying the dye linker moieties, we have sought to evaluate the thermodynamic parameters for intramolecular H-type dye−dye association and the structures of these dimers. We now present data from a series of homodoubly labeled NorFES derivatives that differ by the addition of one or two 6-aminohexanoic acids to the peptide backbone. By comparing absorption and fluorescence propertie...

Recently, we designed and synthesized a new class of profluorescent protease substrates whose spectral properties fit the exciton model; more specifically, spectra of these polypeptides which were doubly labeled with rhodamines showed blue-shifted absorption peaks and fluorescence quenching, both indicators of H-dimer formation. In the work described here NorFES, an undecapeptide which is cleaved by the serine protease elastase, was homodoubly labeled on opposite sides of its cleavage site 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 in the peptide doubly labeled with tetramethylrhodamine, followed by rhodamine-X, and then (diethylamino)coumarin. In contrast, spectra of NorFES homodoubly labeled with fluorescein, hydroxycoumarin, or pyrene do not exhibit exciton bands. These data...