Exploring the heat-induced structural changes of β-lactoglobulin -linoleic acid complex by fluorescence spectroscopy and molecular modeling techniques

Abstract

Linoleic acid (LA) is the precursor of bioactive oxidized linoleic acid metabolites and arachidonic acid, therefore is essential for human growth and plays an important role in good health in general. Because of the low water solubility and sensitivity to oxidation, new ways of LA delivery without compromising the sensory attributes of the enriched products are to be identified. The major whey protein, β-lactoglobulin (β-Lg), is a natural carrier for hydrophobic molecules. The thermal induced changes of the β-Lg-LA complex were investigated in the temperature range from 25 to 85 °C using fluorescence spectroscopy techniques in combination with molecular modeling study and the results were compared with those obtained for β-Lg. Experimental results indicated that, regardless of LA binding, the polypeptide chain rearrangements at temperatures higher than 75 °C lead to higher exposure of hydrophobic residues causing the increase of fluorescence intensity. Phase diagram indicated an all or none transition between two conformations. The LA surface involved in the interaction with β-Lg was about 497 Ǻ(2), indicating a good affinity between those two components even at high temperatures. Results obtained in this study provide important details about heat-induced changes in the conformation of β-Lg-LA complex. The thermal treatment at high temperature does not affect the LA binding and carrier functions of β-Lg.

Keywords: Fluorescence spectroscopy; Linoleic acid; Molecular modeling; Structural changes; β-lactoglobulin.

Fig. 1

Phase diagram analysis of heat-induced conformational changes of β-Lg (grey diamonds) and β-Lg-LA complex (black squares) based on intrinsic fluorescence intensity values measured at wavelengths 320 and 365 nm. The temperature values are indicated in the vicinity of the corresponding symbol. Three independent tests were carried out in each case and SD was lower than 2.5 %

Fig. 2

The increase in Trp (a) and Tyr (b) fluorescence of β-Lg (grey) and β-Lg-LA complex (black) as a function of temperature. The excitation wavelength was 292 and 274 nm respectively. Three independent tests were carried out in each case and SD was lower than 3.5 %. Inset: The emission spectrum of un-treated β-Lg (grey) and β-Lg-LA complex (black)

Fig. 3

Heat-induced structural changes of β-Lg solutions (a) and β-Lg-LA complex (b) at different temperature monitored by ANS fluorescence intensity. Three independent tests were carried out in each case and SD was lower than 3.5 %

Fig. 4

Synchronous fluorescence spectra at ∆λ of 60 nm for β-Lg (a) and β-Lg-LA (b). Three independent tests were carried out in each case and SD was lower than 3.0 %

Fig. 5

Synchronous fluorescence spectra at ∆λ of 15 nm for β-Lg (a) and β-Lg-LA (b). Three independent tests were carried out in each case and SD was lower than 3.0 %

Fig. 6

Details on the β-Lg – LA model equilibrated at 80 °C. a The protein is represented in QuickSurf style, the LA molecule (blue) in Licorice style, and the aminoacid residues directly involved in the interaction with LA (violet) in CPK style using VMD software. b Comparison of LA molecules equilibrated at 25 °C (black) and at 80 °C (blue) by superimposing the C1 atoms of the two molecules