Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates

The neutrophil serine proteases (NSPs) elastase, proteinase 3 and cathepsin G are multifunctional proteases involved in pathogen destruction and the modulation of inflammatory processes. A fraction of secreted NSPs remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities on neutrophil surfaces using highly sensitive Abz-peptidyl-EDDnp fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. We describe FRET substrate synthesis, neutrophil purification and handling, and kinetic experiments on quiescent and activated cells. These are used to measure subnanomolar concentrations of membrane-bound or free NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids like expectorations and bronchoalveolar lavages. the whole procedure, including neutrophil purification and kinetic measurements, can be done in 4-5 h and should not be longer because of the lifetime of neutrophils. Using this protocol will help identify the contributions of individual NSPs to the development of inflammatory diseases and may reveal these proteases to be targets for therapeutic inhibitors.Alexander von Humboldt FoundationGerman Research CouncilVaincre la MucoviscidoseFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Proteases & Vectorisat Pulm Fac Med, INSERM, U618, F-37032 Tours, FranceMax Planck Inst Neurobiol, Dept Neuroimmunol, D-82152 Planegg Martinsried, GermanyUniv Tours, F-37032 Tours, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-0404420 São Paulo, BrazilINSERM, U921, F-37032 Tours, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-0404420 São Paulo, BrazilWeb of Scienc