Effect of Urea upon the Physical Properties of β-Lactoglobulins A and B

Physical alteration o£ fl-lactoglobulin genetic variants A and B was investigated by polyacrylamide gel electrophoresis, Sephadex gel filtration, and nltracentrifugation. fl-Lactoglobulins A and B both exhibited a number of zones on urea polyacrylamide gel electrophoresis.Mercaptoethanol treatment o£ the protein prior to m'ea polyacrylamide gel electrophoresis did not improve the resolution of filactoglobulin or the total whey proteins; however, N-ethyhnaleimide treatment stabilized the fl-lactoglobulln molecule against urea alteration of polyacrylamide gel electrophoresis and Sephadex gel filtration properties.Gel filtration results with Sephadex G-100 indicated that higher urea concentrations caused greater amounts of protein alteration and that these changes were not entirely reversed upon exhaustive dialysis to remove urea.Ultracentrifugation results indicated that the irreversible alteration of fl-lactoglobulin is probably not due to dissociation or aggregation of the protein molecule.The results are in agreement with the concept that urea causes unfolding of the fl-laetoglobulin molecule, which is accompanied by intramolecular disulfide interchange reactions that stabilize the resulting protein conformation upon removM of urea.Urea denaturation of fl-lactoglobulin has been studied by optical rotation and optical rotation dispersion (7, 9, 17, 18), solubility at the isoelectric point (7, 9), ferricyanide reduction and availability of certain side groups on the protein (10), and more recently by the solvent perturbation technique of difference spectroscopy (8), as well as by thermodynamic energy relations (17).Polyacrylamide gel electrophoresis has also been used to demonstrate denaturation of fi-Iactoglobulin in alkaline pH solutions (1, 12).