Effect of heat treatment on the circular dichroism spectra of bovine beta-lactoglobulin A, B, and C

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated in phosphate buffer at temperatures between 40 and 94 degrees C for 10 min, c ooled, and analyzed using near-UV and far-UV circular dichroism (CD). The d ecrease in near-UV CD intensity at 293 nm (Delta epsilon(293)) could be ana lyzed in terms of a two-state model, and the stability was beta-Lg C > beta -Lg A > beta-Lg B on the basis of the midpoint temperatures for samples hea ted at pH 6.7 and 7.4. However, the slopes of the curves at the midpoint te mperature for variant A were generally less than those for beta-Lg B and be ta-Lg C, indicating that the substitution of Val (beta-Lg A) for Ala (beta- Lg B or beta-Lg C) at position 118 had altered the entropic contribution to unfolding of the protein. The changes in CD at 270 nm (Delta epsilon(270)) , an index of significant alteration to disulfide bond dihedral angles, occ urred at higher temperatures than those for the Delta epsilon(293) results. The far-UV CD showed some small changes as a consequence of heat treatment , and the shifts at 205 nm ([theta](205)) fitted a two-state model. Plottin g the changes in both Delta epsilon(293) and [theta](205) against the loss of nativelike and sodium dodecyl sulfate-monomeric protein (assessed by pol yacrylamide gel electrophoresis) showed a strong 1:1 relationship between D elta epsilon(293) or [theta](205) and the loss of nativelike beta-Lg. These results indicated that the initial irreversible stage in the heat-induced aggregation of beta-Lg (nativelike monomer to unfolded monomer) altered the chirality of the environment of Trp(19) and modified the secondary structu re of beta-Lg slightly. The differences in the behavior of variants A-C wer e explicable on the basis of generalized electrostatic and hydrophobicity e ffects as well as specific amino acid effects.