Conformational changes of β-lactoglobulin in sodium bis(2-etliylhexyl) sulfosuccinate reverse micelles: A fluorescence and CD study

Abstract

The effect of β-lactoglobulin encapsulation in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles on the environment of protein and on Trp was analysed at different water contents (ω0). CD data underlined the distortion of the β-sheet and a less constrained tertiary structure as the ω0 increased, in agreement with a concomitant red shift and a decrease in the signal intensity obtained in steady-state fluorescence measurements. Fluorescence lifetimes, evaluated by biexponential analysis, were τ1 = 1.28 ns and τ2 = 3.36 ns in neutral water. In reverse micelles, decay-associated spectra indicated the occurrence of important environmental changes associated with ω0. Bimolecular fluorescence quenching by CCl4 and acrylamide was employed to analyse alterations in the accessibility of the two Trp residues in β-lactoglobulin, induced by changes in ω 0. The average bimolecular quenching constant 〈k qCCl4〉 was found not to depend on ω 0, confirming the insolubility of this quencher in the aqueous interface, while 〈kqacrylamide〉 increases with ω 0. The drastic decrease with ω0 of kq, associated with the longest lifetime, kq2CCl4, comparatively to the increase of kq2acrylamide, emphasizes the location of β-lactoglobulin in the aqueous interfacial region especially at ω0 ≥ 10. The fact that k q2acrylamide (ω0 = 30) ≫ k q2acrylamide (water) also confirms the important conformational changes of encapsulated β-lactoglobulin.