CHARACTERIZATION OF FLUORESCENCE QUENCHING IN BIFLUOROPHORIC PROTEASESUBSTRATES

NorFES is a relatively rigid, bent undecapeptide which contains an ami no acid sequence that is recognized by the serine protease elastase (A spAlaIleProNle down arrow SerIleProLysGlyTyr (down arrow indicates the primary cleavage site)). Covalent attachment of a fluorophore on each side of NorFES's elastase cleavage site enables one to use a change o f fluorescence intensity as a measure of enzymatic activity, In this s tudy two bichromophoric NorFES derivatives, D-NorFES-A and D-NorFES-D, were prepared in which D (donor) was tetramethylrhodamine and A (acce ptor) was rhodamine-X, two chromophores with characteristics suitable for energy transfer. Absorption and fluorescence spectra were obtained with both the intact and cleaved homodoubly, heterodoubly and singly labeled derivatives. It was found that both the home and hetero doubly -labeled derivatives form ground-state complexes which exhibit exciton bands. The hetero labeled derivative exhibits little or no resonance energy transfer. Spectral measurements were also done in urea, which p artially disrupts ground-state dimers. (C) 1997 Elsevier Science B.V.