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 0.3mgmL-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 1mgmL-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. Graphical abstract.
Highlights
Enzymatic studies were assayed in vitro, in dilute conditions, without considering the complexity of real in vivo conditions such as the cellular environment
Hyaluronidase activity was investigated in the absence and in the presence of Polyethylene glycol (PEG) 6000 using the conventional neocuproine spectrophotometry technique as well as capillary electrophoresis (CE)
CE allows the separation and the quantitative detection of different products obtained after the hyaluronic acid (HA) hydrolysis leading to a better understanding of bovine testes hyaluronidase (BTH) activity in a crowded environment and in the presence of inhibitors
Summary
Enzymatic studies were assayed in vitro, in dilute conditions, without considering the complexity of real in vivo conditions such as the cellular environment. The activity of an enzyme in such crowded media can be very different in comparison to dilute conditions [3,4,5]. This may be evaluated experimentally by using concentrated solutions of model “crowding agents” such as polyethylene glycol (PEG), dextran, Ficoll (branched carbohydrate derivative), or inert proteins [5]. Most of the enzymatic studies carried out in vitro under macromolecular crowding (MC) conditions have been monitored either by UV-visible spectroscopy, fluorescence, or far UV-circular dichroism spectroscopy [6,7,8,9]. Some other studies have used isothermal titration calorimetry and turbidimetric methods to study the effect of crowders on enzymatic activity [10, 11]
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