title: Assaying human neutrophil elastase activity by capillary zone electrophoresis combined with laser-induced fluorescence.
abstract: Skin aging is a progressive process determining the ultimate skin appearance. Human neutrophil elastase (HNE) has been shown to play an important role in the degradation of the extracellular matrix. In order to assay HNE kinetics, a novel online capillary zone electrophoresis (CZE) assay has been developed in this study for the determination of the maximum velocity (Vmax) and of the Michaelis-Menten constant (Km) of HNE regarding several potential substrates. These assays are based on short-end injection to shorten analysis time, on transverse diffusion of laminar flow profiles (TDLFP) for in-capillary reactant mixing, and on UV or laser-induced fluorescence (LIF) detection. Kinetic constants for a referenced peptidic substrate were determined using not only online assays but also offline (pre-capillary) mode. The results obtained were cross compared and compared to the literature in order to validate the developed assays. The hydrolysis of three new potential fluorogenic substrates by HNE was also monitored. Two new peptidic substrates for HNE were identified through this study. Km values of these novel substrates were successfully determined using online CZE assay (Km ∼0.07mM). This value was in the same order of magnitude of that of the referenced substrate despite the presence of the labeling group 5-carboxyfluorescein (5-FAM). HNE activity has never been assessed using online CZE-based assay, neither with UV nor with LIF detection. The developed assay conducted with the new labeled substrates is particularly sensitive (LOQ of few nM), does not require the presence of micelles in the BGE (which is the case for the reference substrate) and only necessitates few nanoliters of reactants making it particularly adapted for screening studies.
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Syntia
authors.initials: S
authors.lastname: Fayad
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France. Electronic address: reine.nehme@univ-orleans.fr.
authors.forename: Reine
authors.initials: R
authors.lastname: Nehmé
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Lafite
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Morin
mesh_terms: D004058:Diffusion; D019075:Electrophoresis, Capillary / Q000379:methods; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D007834:Lasers; D019272:Leukocyte Elastase / Q000032:analysis*; D010455:Peptides / Q000032:analysis; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D019272:Leukocyte Elastase
keywords: Enzyme kinetics; Human neutrophil elastase; Laser-induced fluorescence; Skin aging; Transverse diffusion of laminar flow profiles