Polyethylene glycol crowding effect on hyaluronidase activity monitored by capillary electrophoresis
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RESEARCH PAPER
Polyethylene glycol crowding effect on hyaluronidase activity monitored by capillary electrophoresis Rouba Nasreddine 1 & Lucija Orlic 1,2 & Ghassan Al Hamoui Dit Banni 1 & Syntia Fayad 1,3 & Axel Marchal 3 & Francesco Piazza 2 & Chrystel Lopin-Bon 1 & Josef Hamacek 2 & Reine Nehmé 1 Received: 3 March 2020 / Revised: 7 April 2020 / Accepted: 10 April 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract To mimic the activity of hyaluronidase in natural environment, the hydrolysis of hyaluronic acid (HA) by hyaluronidase was investigated for the first time in the presence of crowding agents using capillary electrophoresis (CE) as a simple and reliable technique for conducting enzymatic assay. Polyethylene glycol (PEG) 6000 was selected as a model crowder and the hyaluronic acid degradation catalyzed by bovine testes hyaluronidase (BTH) was carried out at different PEG concentrations (0%, 10%, and 17%). After optimization of the CE analytical method and enzymatic assay, the degradation products were monitored at different HA concentrations. At 10% of PEG and 0.3 mg mL−1 of HA, the activity of the enzyme was significantly reduced showing inconvenient interactions of PEG with the hyaluronidase blocking the release of hydrolysis products. A similar reduction of hyaluronidase activity was observed at 1 mg mL−1 of HA due to the presumable formation of the BTH-substrate complex. The experimental curves obtained by CE also evidence that the overall kinetics are governed by the hydrolysis of hexasaccharide intermediates. Finally, the effect of PEG on hyaluronidase activity was evaluated in the presence of natural or synthetic inhibitors. Our results show a significant difference of the inhibitors’ affinity toward hyaluronidase in the presence of PEG. Surprisingly, the presence of the crowding agent results in a loss of the inhibition effect of small polycyclic inhibitors, while larger charged inhibitors were less affected. In this work, CE analyses confirm the importance of mimicking the cellular environment for the discovery and development of reliable inhibitors.
Keywords Capillary electrophoresis . Crowding . Hyaluronidase activity . Inhibitors . Polysaccharide Abbreviations BGE Background electrolyte BTH Bovine testes hyaluronidase
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Rouba Nasreddine and Lucija Orlic contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02659-9) contains supplementary material, which is available to authorized users. * Josef Hamacek [email protected] * Reine Nehmé [email protected] 1
Institut de Chimie Organique et Analytique (ICOA), CNRS FR 2708 - UMR 7311, Université d’Orléans, 45067 Orléans, France
2
Centre de Biophysique Moléculaire, CNRS UPR 4301, Université d’Orléans - CNRS, 45071 Orléans, France
3
Unité de recherche Œnologie, USC 1366 INRA, Université de Bordeaux, ISVV, EA 5477, 33882 Villenave d’Ornon, France
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