Thermodynamic analysis of ANS binding to partially unfolded α‐lactalbumin: correlation of endothermic to exothermic changeover with formation of authentic molten globules. Issue 9 (6th April 2016)

• Record Type: Journal Article
• Authors: Kim, Ki Hyung; Yun, Soi; Mok, K. H.; Lee, E. K.
• Is Part Of: Journal of molecular recognition. Volume 29:Issue 9(2016)
• Page Start: 446
• Page End: 451
• Publication Date: 2016-04-06
• Subjects: bovine α‐lactalbumin -- ANS -- binding thermodynamics -- fluorescence spectroscopy -- ITC -- molten globule; Molecular recognition -- Periodicals; Models, Molecular -- Periodicals; Molecular Conformation -- Periodicals; Molecular Sequence Data -- Periodicals; Molecular Structure -- Periodicals; Carrier Proteins -- Periodicals; 572.8
• DOI: 10.1002/jmr.2543
• Languages: English
• ISSNs: 0952-3499

Abstract

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. Abstract : The conspicuous mirror image of the isothermal titration calorimetry titration curves for bovine α‐LA‐8‐anilino‐1‐naphthalene sulfonic acid binding indicates the thermodynamic changeover from endothermic at 20°C (a) to exothermic binding at 40°C (b), which might correlate with the increased formation and population of molten globules of bovine α‐LA at 40°C.