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Competitive binding of fatty acids and the fluorescent probe 1-8-anilinonaphthalene sulfonate to bovine ß-lactoglobulin

¹ Dipartimento di Fisica, Università degli Studi di Milano-Bicocca, 20126 Milano, Italy
² Istituto per lo Studio delle Macromolecole, Consiglio Nazionale delle Ricerche (CNR), 20131 Milano, Italy
³ Dipartimento Scientifico e Tecnologico, Università di Verona, Ca' Vignal 1, 37134 Verona, Italy
⁴ Istituto Nazionale per la Fisica della Materia, INFM, UdR Milano-Bicocca, 20126 Milano, Italy

Reprint requests to: Giancarlo Baldini, Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, 20126 Milano, Italy; e-mail: giancarlo.baldini{at}mib.infn.it; fax: 39-0264482894.

The use of spectroscopy in the study of fatty acids binding to bovine ß-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS–BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

Keywords: Fatty acid; ß-lactoglobulin; ANS; time resolved fluorescence; binding; docking

Abbreviations: BLG, bovine ß-lactoglobulin • NMR, nuclear magnetic resonance • ANS, 1-8-anilinonaphthalene sulfonate • DMSO, dimethylsulfoxide • MIF, molecular interaction field • PDB, Protein Data Bank

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