Effect of heat treatment on bovine beta-lactoglobulin A, B, and C exploredusing thiol availability and fluorescence

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated at temperatures between about 40 and 94 degrees C for 10 min, cooled, and ana lyzed using Trp fluorescence and extrinsic fluorescence spectra of the prob e 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-d ithiobis-(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thio l availability results showed a 1:1 relationship with the loss of nativelik e but not SDS-monomeric protein, as determined by PAGE analysis. The normal ized Trp emission intensity results did not show a comparable 1:1 relations hip with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state mo dels, and the midpoint temperatures (T-mid) for the proteins were generally beta-Lg C > beta-Lg A > beta-Lg B, and the slopes at the midpoint temperat ures for the A variant were generally less than those for the B and C varia nts indicating that beta-Lg A may denature by a different mechanism from th at of beta-Lg B or beta-Lg C. The T-mid parameters derived from the ANS flu orescence intensity results were similar to those for thiol availability an d both were lower than the T-mid values for Trp emission intensity showing that creation of an ANS binding site on a beta-Lg molecule was linked to th e irreversible exposure of a thiol group and the loss of native beta-Lg but preceded the decrease in Trp(61) fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants invo lved the creation of a destabilizing cavity by the Val(118)Ala (A --> B) su bstitution and the changed charge distribution within the CD loop caused by the Asp(64)Gly (A --> B) substitution.