Polyethylene glycol crowding effect on hyaluronidase activity monitored by capillary electrophoresis

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.

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Abbreviations

BGE:

Background electrolyte

BTH:

Bovine testes hyaluronidase

CPA:

Corrected peak area

CS-A:

Biotinylated 4-sulfated chondroitin tetrasaccharides

CS-C:

Biotinylated 6-sulfated chondroitin tetrasaccharides

Dec:

Decasaccharide

Dod:

Dodecasaccharide

ECM:

Extracellular matrix

EGCG:

Epigallocatechin gallate

HA:

Hyaluronic acid

Hdec:

Hexadecasaccharide

Hex:

Hexasaccharide

IB:

Incubation buffer

MC:

Macromolecular crowding

Oct:

Octasaccharide

QTT-I:

Quercotriterpenoside-I

Tdec:

Tetradecasaccharide

Tet:

Tetrasaccharide

μeph :

Electrophoretic mobility

DIM:

Dimeric

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Funding

This work has been financially supported by the University of Orléans (France), the CNRS (Centre National de la Recherche Scientifique, France), and the Labex SynOrg (ANR-11-LABX-0029).

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Nasreddine, R., Orlic, L., Al Hamoui Dit Banni, G. et al. Polyethylene glycol crowding effect on hyaluronidase activity monitored by capillary electrophoresis. Anal Bioanal Chem 412, 4195–4207 (2020). https://doi.org/10.1007/s00216-020-02659-9

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Keywords

  • Capillary electrophoresis
  • Crowding
  • Hyaluronidase activity
  • Inhibitors
  • Polysaccharide