title: Hyaluronidase reaction kinetics evaluated by capillary electrophoresis with UV and high-resolution mass spectrometry (HRMS) detection.
abstract: The biology of hyaluronidase activity on age related turnover of the hyaluronic acid (HA) in skin dermis and epidermis has not been established. Elucidation of this phenomenon enables discovery of novel compounds for skin health. As a simple and green technique, capillary electrophoresis (CE) was used for the first time for the determination of the kinetic constants (Km,Vmaxand IC50) of the enzymatic degradation of HA. Reaction products were identified using CE/high-resolution mass spectrometry (HRMS) after appropriate optimization. Best results in terms of signal sensitivity were obtained using 10 mM ammonium acetate (pH 9.0) BGE, a sheath liquid composed of methanol-water (80:20, v/v) with 0.02% (v/v) formic acid at 10 μL min-1and an ESI voltage at -4 kV. Kmand Vmaxwere determined (n = 3) using CE/UV at 200 nm as 0.24 ± 0.02 mg mL-1and 150.4 ± 0.1 nM s-1, respectively. They were also successfully obtained by CE/HRMS (n = 3) with Kmof 0.49 ± 0.02 mg mL-1and Vmaxof 155.7 ± 0.2 nM s-1. IC50of a standard natural inhibitor, epigallocatechin gallate, was also determined by CE-UV/HRMS. Kinetic constant values obtained by CE compared well with literature which validated the developed CE-based assay. In addition, the activity of homemade tetrasaccharides of biotinylated chondroitin sulfate CS-A or CS-C (4- or 6- sulfated in a homogeneous or heterogeneous way) on the hydrolysis reaction of hyaluronidase was evaluated. Hyaluronidase was mostly dose-dependently inhibited by CS-A tetrasaccharides sulfated in a homogeneous way. Two trisaccharides from truncated linkage region of proteoglycans were also tested as inhibitors or activators. CE-based assay showed that even a small modification of one hydroxyl group changes the influence on hyaluronidase activity. CE-based assay can be used for the screening of natural and synthetic inhibitors of hyaluronidase activity for cosmetic and therapeutic applications.
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: Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czechia.
authors.forename: Monika
authors.initials: M
authors.lastname: Langmajerová
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ayela
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France; CNRS, CBM, UPR 4301, Université d'Orléans, F-45071, Orléans, France.
authors.forename: Cyril
authors.initials: C
authors.lastname: Colas
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Benoit
authors.initials: B
authors.lastname: Maunit
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Jacquinet
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Aude
authors.initials: A
authors.lastname: Vibert
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Chrystel
authors.initials: C
authors.lastname: Lopin-Bon
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czechia.
authors.forename: Glatz
authors.initials: G
authors.lastname: Zdeněk
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: D019075:Electrophoresis, Capillary; D006821:Hyaluronoglucosaminidase / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry
chemical_list: D006821:Hyaluronoglucosaminidase
keywords: Capillary electrophoresis; Chondroitin sulfate; Enzyme; High-resolution mass spectrometry; Hyaluronidase