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Studying Gastric Lipase Adsorption Onto Phospholipid Monolayers by Surface Tensiometry, Ellipsometry, and Atomic Force Microscopy

Abstract : The access to kinetic parameters of lipolytic enzyme adsorption onto lipids is essential for a better understanding of the overall catalytic process carried out by these interfacial enzymes. Gastric lipase, for instance, shows an apparent optimum activity on triglycerides (TAG) at acidic pH, which is controlled by its pH-dependent adsorption at lipid–water interfaces. Since gastric lipase acts on TAG droplets covered by phospholipids, but does not hydrolyze these lipids, phospholipid monolayers spread at the air–water interfaces can be used as biomimetic interfaces to study lipase adsorption and penetration through the phospholipid layer, independently from the catalytic activity. The adsorption of recombinant dog gastric lipase (rDGL) onto 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) monolayers can be monitored by surface tensiometry at various enzyme concentrations, pHs, and surface pressures (Π). These experimental data and the use of Langmuir adsorption isotherm and Verger–de Haas’ lipase kinetics models further allow estimating various parameters including the adsorption equilibrium constant (KAds), the interfacial concentration Γ E * , the molar fraction Φ E * % , mol % (ΦE*(%), mol%), and the molecular area A E * of rDGL adsorbed onto the DLPC monolayer under various conditions. Additional insight into rDGL adsorption/insertion on phospholipid monolayers can be obtained by combining ellipsometry, Langmuir–Blodgett film transfer, and atomic force microscopy. When using multicomponent phospholipid monolayers with phase separation, these techniques allow to visualizing how rDGL preferentially partitions toward liquid expanded phase and at phase boundaries, gets adsorbed at various levels of insertion and impacts on the lateral organization of lipids.
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https://hal-univ-rennes1.archives-ouvertes.fr/hal-01484629
Contributor : Laurent Jonchère <>
Submitted on : Tuesday, March 7, 2017 - 2:56:35 PM
Last modification on : Saturday, October 3, 2020 - 3:13:48 AM

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A. Bénarouche, L. Sams, C. Bourlieu, V. Vié, V. Point, et al.. Studying Gastric Lipase Adsorption Onto Phospholipid Monolayers by Surface Tensiometry, Ellipsometry, and Atomic Force Microscopy. Gelb, Michael H. Methods in Enzymology, 583 (Chapter Eleven), Academic Press, pp.255-278, 2017, Enzymology at the Membrane Interface: Interfacial Enzymology and Protein-Membrane Binding, 978-0-12-809419-8. ⟨10.1016/bs.mie.2016.09.039⟩. ⟨hal-01484629⟩

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