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Pressure denaturation and aggregation of β-lactoglobulin studied by intrinsic fluorescence depolarization, Rayleigh scattering, radiationless energy transfer and hydrophobic fluoroprobing

Published online by Cambridge University Press:  01 November 1999

HENRIK STAPELFELDT
Affiliation:
Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Present address: Institute of Food Research and Nutrition, Danish Veterinary and Food Administration, Søndervang 4, DK-4100 Ringsted, Denmark.
LEIF H. SKIBSTED
Affiliation:
Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark

Abstract

β-Lactoglobulin (β-lg) in aqueous solution under pressure showed a marked depolarization of intrinsic fluorescence assigned to a gradually increased rotational diffusion of tryptophyl moieties in pressure-unfolded states. The corresponding change in anisotropy provided a new and more accurate method for determining denaturation volume which, for β-lg in neutral aqueous solution with ionic strength 0·16 (NaCl) at 25 °C, was ΔV°=−73 (SE 3) ml mol−1, corresponding to half denaturation at 123 MPa. The pressure unfolding led to exposure of hydrophobic regions to the protein–water interface that could be probed by fluorescence intensity of a β-lg–1-anilinonaphthalene-8-sulphonic acid (ANS) complex with 1:1 stoichiometry, as determined by Job's method of continuous variation. The unfolding of β-lg impaired the binding capacity of the inner calyx, with a reduction in binding capacity of 50% at 50 MPa, as shown by decreasing cis- parinaric acid fluorescence, decreasing anisotropy and decreasing radiationless energy transfer from tryptophans to this probe with increasing pressure. The pressure-induced reversible exposure of hydrophobic groups to the protein–water interface may, at least partly, explain the initial aggregation reactions, evident from increased Rayleigh scattering from ∼50 MPa, prior to irreversible pressure-induced gel formation of β-lg. Using results from this and previous studies, we propose a three step pressure denaturation model for β-lg for neutral solution at ambient temperature, including an initial pressure-melted state (up to 50 MPa) with partial collapse of the inner calyx and solvent exposure of the free thiol group, followed by a reversible denaturation with exposure of hydrophobic regions (half denaturation at 123 MPa) and with irreversible denaturation with thiol–disulphide exchange becoming increasingly important at higher pressures. Effects of pressure on β-lg, as measured by fluorescence depolarization, were found for the reversible denaturation steps to be similar to the effects of chemical denaturants but different with respect to shift in ANS emission maxima.

Type
Research Article
Copyright
Proprietors of Journal of Dairy Research 1999

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