INTRAMOLECULAR RESONANCE DIPOLE-DIPOLE INTERACTIONS IN A PROFLUORESCENT PROTEASE SUBSTRATE

In this study NorFES, an undecapeptide containing an amino acid sequen ce recognized by the serine protease elastase, was covalently labeled with two xanthenes, one on each side of its cleavage site, to serve as a tool for examination of intramolecular resonance dipole-dipole inte ractions. To this end using all possible combinations from the group o f xanthenes including fluorescein, tetramethylrhodamine, and rhodamine -X, three heterobichromophoric and three homobichromophoric NorFES der ivatives were synthesized; their absorption and fluorescence spectra w ere measured both before and after cleavage by elastase. In the hetero bichromophoric substrates the fluorescence of the fluorophore that wou ld be the nominal donor in a Forster model system was quenched. Since the fluorescence intensity of the nominal acceptor in these substrates was also decreased, these data were not consistent with the Forster m odel. Rather, spectra for all six doubly labeled peptides could be exp lained by delocalization of excitation over each substrate's two fluor ophores. Thus, by taking into account dipole-dipole interactions betwe en two dyes placed in close proximity to each other, the spectral prop erties observed could not be ascribed to the monomeric components but were the unique optical signature of each ground-state dimer.