Profluorescent protease substrates: intramolecular dimers described by the exciton model.
Xanthene dyes are known to form dimers with spectral characteristics that have been interpreted in terms of exciton theory. A unique aspect of H-type dimers is the fluorescence quenching that accompanies their formation. Using the principles of exciton theory as a guide, a series of protease substrates was synthesized with a xanthene dye on each side of the cleavage site. To bring the attached dyes into spatial proximity to form a dimer, the molecular design included structure determinant regions in the amino acid sequence. In addition, chromophores were chosen such that changes in absorption spectra indicative of exciton splitting were anticipated. Cleavage of the peptides by a protease resulted in disruption of the dimers and indeed significant absorption spectral changes were observed. Furthermore, substrate cleavage was accompanied by at least an order of magnitude increase in fluorescence intensity. This has allowed determination of intracellular elastase activity using a fluorescence microscope equipped with standard optics.
Packard
B Z
BZ
OncoImmunin, Inc., College Park, MD 20742, USA.
Toptygin
D D
DD
Komoriya
A
A
Brand
L
L
D000595:Amino Acid Sequence; D019281:Dimerization; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D005456:Fluorescent Dyes; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism; D011487:Protein Conformation; D012235:Rhodamines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014966:Xanthenes
C101964:D-NorFES-D; D005456:Fluorescent Dyes; D009842:Oligopeptides; D012235:Rhodamines; D014966:Xanthenes; D010450:Endopeptidases; D010196:Pancreatic Elastase