title: Three wavelength substrate system of neutrophil serine proteinases.
abstract: Neutrophil serine proteases, including elastase, proteinase 3, and cathepsin G, are closely related enzymes stored in similar amounts in azurophil granules and released at the same time from triggered neutrophils at inflammatory sites. We have synthesized new fluorescence resonance energy transfer (FRET) substrates with different fluorescence donor-acceptor pairs that allow all three proteases to be quantified at the same time and in the same reaction mixture. This was made possible because the fluorescence emission spectra of the fluorescence donors do not overlap and because the values of the specificity constants were in the same range. Thus, similar activities of proteases can be measured with the same sensitivity. In addition, these substrates contain an N-terminal 2-(2-(2-aminoethoxy)ethoxy)acetic acid (PEG) moiety that makes them cell permeable. Using the mixture of these selected substrates, we were able to detect the neutrophil serine protease (NSP) activity on the activated neutrophil membrane and in the neutrophil lysate in a single measurement. Also, using the substrate mixture, we were in a position to efficiently determine NSP activity in human serum of healthy individuals and patients with diagnosed Wegener disease or microscopic polyangiitis.
authors: Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland.
authors: Magdalena
authors: M
authors: Wysocka
authors: Adam
authors: A
authors: Lesner
authors: Natalia
authors: N
authors: Gruba
authors: Brice
authors: B
authors: Korkmaz
authors: Francis
authors: F
authors: Gauthier
authors: Mizuki
authors: M
authors: Kitamatsu
authors: Anna
authors: A
authors: Łęgowska
authors: Krzysztof
authors: K
authors: Rolka
mesh_terms: D000818:Animals; D002417:Cattle; D015195:Drug Design; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D009504:Neutrophils / Q000201:enzymology*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D057057:Serine Proteases / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D057057:Serine Proteases