title: Measurement of neutrophil elastase, proteinase 3, and cathepsin G activities using intramolecularly quenched fluorogenic substrates.
abstract: Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases, large quantities of which are stored in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to degrade engulfed microorganisms inside phagolysosomes. Active forms of these proteases are also externalized during neutrophil activation at inflammatory sites, thus helping to regulate inflammatory and immune responses. A fraction of secreted neutrophil serine proteases (NSPs) remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities using sensitive ortho-aminobenzoyl-peptidyl-N-(2,4-dinitrophenyl) ethylenediamine fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. These are used to measure subnanomolar concentrations of free or membrane-bound NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids. We describe the synthesis of FRET substrate, neutrophil purification, and kinetic experiments on activated neutrophils. The protocol for measuring NSP activity on the surface of activated neutrophils can be adapted to measure NSP activities in whole biological fluids. Such data clarify the contributions of individual NSPs to the development of inflammatory diseases. Ultimately, these proteases may be shown to be targets for therapeutic inhibitors.
authors.affiliation: INSERM U-618 Protéases et Vectorisation Pulmonaires, Tours, and Université François Rabelais, Tours, 37032, France.
authors.forename: Brice
authors.initials: B
authors.lastname: Korkmaz
authors.forename: Sylvie
authors.initials: S
authors.lastname: Attucci
authors.forename: Christophe
authors.initials: C
authors.lastname: Epinette
authors.forename: Elodie
authors.initials: E
authors.lastname: Pitois
authors.forename: Marie-Lise
authors.initials: ML
authors.lastname: Jourdan
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Francis
authors.initials: F
authors.lastname: Gauthier
mesh_terms: D056649:Cathepsin G / Q000378:metabolism*; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism*; D053146:Myeloblastin / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D056649:Cathepsin G; D019272:Leukocyte Elastase; D053146:Myeloblastin