title: Thermodynamic analysis of ANS binding to partially unfolded α-lactalbumin: correlation of endothermic to exothermic changeover with formation of authentic molten globules.
abstract: A fluorescent reporter, 8-anilino-1-naphthalene sulfonic acid (ANS), can serve as a reference molecule for conformational transition of a protein because its aromatic carbons have strong affinity with hydrophobic cores of partially unfolded molten globules. Using a typical calcium-binding protein, bovine α-lactalbumin (BLA), as a model protein, we compared the ANS binding thermodynamics to the decalcified (10 mM EDTA treated) apo-BLA at two representative temperatures: 20 and 40 °C. This is because the authentic molten globule is known to form more heavily at an elevated temperature such as 40 °C. Isothermal titration calorimetry experiments revealed that the BLA-ANS interactions at both temperatures were entropy-driven, and the dissociation constants were similar on the order of 10(-4)  M, but there was a dramatic changeover in the binding thermodynamics from endothermic at 20 °C to exothermic at 40 °C. We believe that the higher subpopulation of authentic molten globules at 40 °C than 20 °C would be responsible for the results, which also indicate that weak binding is sufficient to alter the ANS binding mechanisms. We expect that the thermodynamic properties obtained from this study would serve as a useful reference for investigating the binding of other hydrophobic ligands such as oleic acid to apo-BLA, because oleic acid is known to have tumor-selective cytotoxicity when complexed with partially unfolded α-lactalbumin. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Department of Bionano Engineering, Hanyang University - ERICA, Ansan, Korea.
authors.forename: Ki Hyung
authors.initials: KH
authors.lastname: Kim
authors.affiliation: Department of Bionano Engineering, Hanyang University - ERICA, Ansan, Korea.
authors.forename: Soi
authors.initials: S
authors.lastname: Yun
authors.affiliation: Trinity Biomedical Sciences Institute (TBSI), School of Immunology and Biochemistry, Dublin 2, Ireland.|Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), Trinity College, The University of Dublin, Dublin 2, Ireland.
authors.forename: K H
authors.initials: KH
authors.lastname: Mok
authors.affiliation: Department of Bionano Engineering, Hanyang University - ERICA, Ansan, Korea.
authors.forename: E K
authors.initials: EK
authors.lastname: Lee
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D002151:Calorimetry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000378:metabolism*; D019301:Oleic Acid / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D019301:Oleic Acid; D007768:Lactalbumin
keywords: ANS; ITC; binding thermodynamics; bovine α-lactalbumin; fluorescence spectroscopy; molten globule