Assaying human neutrophil elastase activity by capillary zone electrophoresis combined with laser-induced fluorescence.
作者： Syntia Fayad , Reine Nehmé , Pierre Lafite , Philippe Morin
DOI: 10.1016/J.CHROMA.2015.09.084
关键词:
摘要: Skin aging is a progressive process determining the ultimate skin appearance. Human neutrophil elastase (HNE) has been shown to play an important role in degradation of extracellular matrix. In order assay HNE kinetics, novel online capillary zone electrophoresis (CZE) developed this study for determination maximum velocity (Vmax) and Michaelis-Menten constant (Km) regarding several potential substrates. These assays are based on short-end injection shorten analysis time, transverse diffusion laminar flow profiles (TDLFP) in-capillary reactant mixing, UV or laser-induced fluorescence (LIF) detection. Kinetic constants referenced peptidic substrate were determined using not only but also offline (pre-capillary) mode. The results obtained cross compared literature validate assays. hydrolysis three new fluorogenic substrates by was monitored. Two identified through study. Km values these successfully CZE (Km ∼0.07mM). This value same magnitude that despite presence labeling group 5-carboxyfluorescein (5-FAM). activity never assessed CZE-based assay, neither with nor LIF conducted labeled particularly sensitive (LOQ few nM), does require micelles BGE (which case reference substrate) necessitates nanoliters reactants making it adapted screening studies.
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