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

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Abstract
Physical alteration of [beta]-lacto-globulin genetic variants A and B was investigated by polyacrylamide gel electrophoresis, Sephadex gel filtration, and ultracentrifugation. [beta]-lactoglobulins A and B both exhibited a number of zones on urea polyacrylamide gel electrophoresis. Mercaptoethanol treatment of the protein prior to urea polyacrylamide gel electrophoresis did not improve the resolution of [beta]-lactoglobulin or the total whey proteins; however, N-ethylmaleimide treatment stabilized the [beta]-lactoglobulin 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 [beta]-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 B-lactoglobulin molecule, which is accompanied by intramolecular disulfide interchange reactions that stabilize the resulting protein conformation upon removal of urea.