Heat Treatment of β-Lactoglobulin:  Structural Changes Studied by Partitioning and Fluorescence

Abstract

Functional properties of whey protein concentrates (WPC) are primarily dependent on the degree of denaturation of beta-lactoglobulin (beta-LG), the major globular whey protein. Irreversible modifications in the tertiary structure and association state of beta-LG after heat treatment were studied by partition in aqueous two-phase systems and fluorescence quenching. Partitioning of preheated beta-LG in two-phase systems containing 5% (w/w) poly(ethylene glycol) and 7% (w/w) dextran, between pH 6.0 and7.0, are appropriately related with the intensity of heat treatment. An increase in the partition coefficient of beta-LG was observed with increasing temperature of heat treatment. On the other hand, fluorescence quenching of beta-LG by acrylamide was used to study the conformational flexibility of the protein at pH values between 4. 0 and 9.0. The values of bimolecular quenching rate constant (k(q)) obtained showed that beta-LG appears to be more flexible at high pH values, while at low pH the protein assumes a more compact form. The efficiency of acrylamide quenching on preheated beta-LG was substantially more pronounced than for the untreated protein. This difference can be ascribed to the presence of unfolded monomers and aggregates of denatured molecules formed after heat treatment, whose tryptophanyl residues are more exposed to the solvent. In conclusion, the results suggest that partition studies in aqueous two-phase systems and fluorescence quenching are very useful tools to detect changes in conformation and aggregation of beta-LG induced by heat treatment.

Interaction of epigallocatechin-3-gallate with β-lactoglobulin: molecular characterization and biological implication

“…In comparison with β-Lg, h-β-Lg has a λ max of 340 nm with a red shift of 6 nm (Fig. 3B) due to the partial loss of the hydrophobicity of the Trp19 environment and stronger intensity because of the decreased quenching of Trp61 (Palazolo et al 2000). Addition of EGCG to h-β-Lg induced changes in λ max somewhat analogous to those obtained with β-Lg but at longer wavelengths λ max ¼ 352 nm at a protein=antioxidant ratio of 1 : 10 ð Þ .…”

Binding of alkylsulfonate ligands to bovine β-lactoglobulin: Effects on protein thermal unfolding

“…(1) below 55°C, b-LG solutions are a mixture of monomers and dimers Aymard et al, 1996;Galani & Apenten, 1999;Qi et al, 1995), (2) between 55 and 90°C, b-LG heating induces irreversible changes in the monomer structure: at least one molten globule-like state and possibly aggregates of low molecular weight were produced (Apenten et al, 2002;Croguennec et al, 2004;Fessas et al, 2001;Manderson et al, 1988;Palazolo et al, 2000;Relkin, 1996), (3) above 90°C, b-LG aggregates of high molecular weight are formed irreversibly.…”

Thermal unfolding of bovine β-lactoglobulin studied by UV spectroscopy and fluorescence quenching

“…This major exposition of Trp residues to the aqueous solvent promotes an increase in the fluorescence quenching of denatured proteins by acrylamide. More flexible is the protein structure and/or the higher is the concentration of unfolded species, the more pronounced is the Stern-Volmer plot (Busti, Scarpeci, Gatti, & Delorenzi, 2002;Moro, Gatti, & Delorenzi, 2001;Palazolo et al, 2000).…”

“…The intrinsic fluorescence of Trp residues is particularly sensitive to their microenvironments and provides an appropriate method to perform this kind of study. The degree of exposure of Trp residues in the b-LG molecule can be evaluated by following the external quenching of the intrinsic protein fluorescence by added solutes (Busti, Scarpeci, Gatti, & Delorenzi, 1999;Palazolo, Rodríguez, Farruggia, Picó , & Delorenzi, 2000). To complement the discussion about the thermal unfolding of b-LG, we used the intrinsic fluorescence quenching by acrylamide to examine the conformational states of the heat-induced species of the protein.…”