The stabilizing role of the disaccharide trehalose on beta-lactoglobulin (BLG) against its chemical denaturation both at native and acidic pH has been explored by means of time-resolved fluorescence of the probe acrylodan covalently bound to the unique free cysteine of BLG. The changes in acrylodan fluorescence lifetime with guanidinium chloride concentration reveal BLG sigmoidal denaturation profiles which depend upon the amount of trehalose in solution. When adding trehalose the transition midpoint shifts towards higher denaturant concentration. This effect has been measured by fitting the data with a two-state model whose parameters indicate that an almost 60% increase in the denaturation free energy is induced independently of trehalose concentrations and pH values. Fluorescence anisotropy measurements performed in the same conditions reveal that the internal dynamics are largely affected by the sugar, which makes the acrylodan environment more rigid, and by the denaturant that acts in the opposite way. The overall rotational diffusion of BLG suggests that trehalose affects the hydrodynamic properties of the solution in the proximity of the protein; tentative mechanisms are discussed.

D'Alfonso, L., Collini, M., Baldini, G. (2003). Trehalose influence on beta-lactoglobulin stability and hydration by time resolved fluorescence. EUROPEAN JOURNAL OF BIOCHEMISTRY, 270(11), 2497-2504 [10.1046/j.1432-1033.2003.03621.x].

Trehalose influence on beta-lactoglobulin stability and hydration by time resolved fluorescence

D'ALFONSO, LAURA;COLLINI, MADDALENA;BALDINI, GIANCARLO
2003

Abstract

The stabilizing role of the disaccharide trehalose on beta-lactoglobulin (BLG) against its chemical denaturation both at native and acidic pH has been explored by means of time-resolved fluorescence of the probe acrylodan covalently bound to the unique free cysteine of BLG. The changes in acrylodan fluorescence lifetime with guanidinium chloride concentration reveal BLG sigmoidal denaturation profiles which depend upon the amount of trehalose in solution. When adding trehalose the transition midpoint shifts towards higher denaturant concentration. This effect has been measured by fitting the data with a two-state model whose parameters indicate that an almost 60% increase in the denaturation free energy is induced independently of trehalose concentrations and pH values. Fluorescence anisotropy measurements performed in the same conditions reveal that the internal dynamics are largely affected by the sugar, which makes the acrylodan environment more rigid, and by the denaturant that acts in the opposite way. The overall rotational diffusion of BLG suggests that trehalose affects the hydrodynamic properties of the solution in the proximity of the protein; tentative mechanisms are discussed.
Articolo in rivista - Articolo scientifico
acrylodan; beta-lactoglobulin; internal dynamics; stability; trehalose
English
giu-2003
270
11
2497
2504
none
D'Alfonso, L., Collini, M., Baldini, G. (2003). Trehalose influence on beta-lactoglobulin stability and hydration by time resolved fluorescence. EUROPEAN JOURNAL OF BIOCHEMISTRY, 270(11), 2497-2504 [10.1046/j.1432-1033.2003.03621.x].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/17209
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