Korkmaz Brice, Attucci Sylvie, Juliano Maria Aparecida, Kalupov Timofey, Jourdan Marie-Lise, Juliano Luiz, Gauthier Francis
Department of Neuroimmunology, Max-Planck Institute of Neurobiology, Planegg-Martinsried D-82152, Germany.
Nat Protoc. 2008;3(6):991-1000. doi: 10.1038/nprot.2008.63.
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.
中性粒细胞丝氨酸蛋白酶（NSPs），即弹性蛋白酶、蛋白酶3和组织蛋白酶G，是参与病原体破坏和炎症过程调节的多功能蛋白酶。一部分分泌的NSPs仍与细胞外质膜结合，并在那里保持酶活性。本方案描述了使用高度敏感的Abz-肽基-EDDnp荧光共振能量转移（FRET）底物，通过荧光分光光度法测量中性粒细胞表面NSPs的活性，该底物能完全区分三种人类NSPs。我们描述了FRET底物的合成、中性粒细胞的纯化和处理，以及对静止和活化细胞的动力学实验。这些实验用于在低结合微孔板中测量亚纳摩尔浓度的膜结合或游离NSPs，并量化痰液和支气管肺泡灌洗等生物体液中单个蛋白酶的活性。整个过程，包括中性粒细胞的纯化和动力学测量，可以在4至5小时内完成，由于中性粒细胞的寿命，不应超过此时间。使用本方案将有助于确定单个NSPs在炎症性疾病发展中的作用，并可能揭示这些蛋白酶可作为治疗性抑制剂的靶点。
