Heat treatment of beta-lactoglobulin: structural changes studied by partitioning and fluorescence.
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.
Palazolo
G
G
Area Fisicoquímica, Departamento de Química-Física, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531 (2000), Rosario, Argentina.
Rodríguez
F
F
Farruggia
B
B
Picó
G
G
Delorenzi
N
N
D005453:Fluorescence; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry*; D011487:Protein Conformation
D007782:Lactoglobulins