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Investigation of lipase-ligand interactions in porcine pancreatic extracts by microscale thermophoresis

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Abstract

The evaluation of binding affinities between large biomolecules and small ligands is challenging and requires highly sensitive techniques. Microscale thermophoresis (MST) is an emerging biophysical technique used to overcome this limitation. This work describes the first MST binding method to evaluate binding affinities of small ligands to lipases from crude porcine pancreatic extracts. The conditions of the MST assay were thoroughly optimized to successfully evaluate the dissociation constant (Kd) between pancreatic lipases (PL) and triterpenoid compounds purified from oakwood. More precisely, the fluorescent labeling of PL (PL*) using RED-NHS dye was achieved via a buffer exchange procedure. The MST buffer was composed of 20 mM NaH2PO4 + 77 mM NaCl (pH 6.6) with 0.05% Triton-X added to efficiently prevent protein aggregation and adsorption, even when using only standard, uncoated MST capillaries. Storage at −20 °C ensured stability of PL* and its fluorescent signal. MST results showed that crude pancreatic extracts were suitable as a source of PL for the evaluation of binding affinities of small ligands. Quercotriterpenoside-I (QTT-I) demonstrated high PL* binding affinity (31 nM) followed by 3-O-galloylbarrinic acid (3-GBA) (500 nM) and bartogenic acid (BA) (1327 nM). To enrich the 50 kDa lipase responsible for the majority of hydrolysis activity in the crude pancreatic extracts, ammonium sulfate precipitation was attempted and its efficiency confirmed using capillary electrophoresis (CE)-based activity assays and HRMS. Moreover, to accurately explain enzyme modulation mechanism, it is imperative to complement binding assays with catalytic activity ones.

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Acknowledgements

The authors would like to thank Dr. B. Claude for her aid and advices in lipase purification experiments, Dr. D. Da Silva for his aid and advices for MS analyses and lipase desalting, F. Coudray for the design and construction of the 3D printed scaffold for the C4D cell utilized in CE assays, and C. Maffre for technical support.

Funding

This work has been financially supported by the Région Centre Val de Loire (PhD fellowship of G. Al Hamoui Dit Banni) and the Labex SynOrg (ANR-11- LABX-0029).

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Authors and Affiliations

  1. Institut de Chimie Organique et Analytique (ICOA), CNRS FR 2708 – UMR 7311, Université d’Orléans, 45067, Orléans, France

    Ghassan Al Hamoui Dit Banni, Rouba Nasreddine, Cyril Colas & Reine Nehmé

  2. Centre de Biophysique Moléculaire, CNRS-Université d’Orléans, UPR 4311, 45071, Orléans CEDEX 2, France

    Cyril Colas

  3. Institut des Sciences de la Vigne et du Vin (ISVV), EA 5477, Unité de recherche Œnologie, USC 1366 INRA, Université de Bordeaux, 33882, Villenave d’Ornon, France

    Syntia Fayad & Axel Marchal

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  1. Ghassan Al Hamoui Dit Banni
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Contributions

G. Al Hamoui Dit Banni and R. Nasreddine planned and performed MST and CE-based enzymatic experiments and analyzed the corresponding data; G. Al Hamoui Dit Banni and C. Colas carried out the HRMS assessment of PL purification; S. Fayad and A. Marchal planned and performed purifications and characterization of oakwood and wine extracts. R. Nehmé managed the project and data analysis. All the authors discussed the results and contributed to the final manuscript.

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Correspondence to Reine Nehmé.

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Al Hamoui Dit Banni, G., Nasreddine, R., Fayad, S. et al. Investigation of lipase-ligand interactions in porcine pancreatic extracts by microscale thermophoresis. Anal Bioanal Chem 413, 3667–3681 (2021). https://doi.org/10.1007/s00216-021-03314-7

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