J. Biol. Chem. 268 : 22414-22419 (1993)

Unfolding/Refolding Studies on Bovine Beta-Lactoglobulin with Monoclonal Antibodies as Probes: Does a Renatured Protein Completely Refold ?

M. Hattori, A. Ametani, Y. Katakura, M. Shimizu and S. Kaminogawa

Department of Agricultural Chemistry,
Faculty of Agriculture,
The University of Tokyo
Tokyo 113, Japan

We investigated whether any local moieties within a protein molecule could completely refold from the denatured state to regain the native conformation. Bovine beta-lactoglobulin (BLG) was denatured in the presence of guanidine hydrochloride (GdnHCl) as the denaturant. Renaturation of the denatured BLG was attempted by dialyzing to remove GdnHCl. The renatured molecules regained the same retinol binding activity as that of native BLG, and physicochemical studies also indicated that refolding of the denatured BLG had been almost completely successful. Local structural differences between the native and renatured BLG molecules were evaluated by using our panel of four anti-BLG monoclonal antibodies (anti BLG mAbs). The structures of the epitope regions in native BLG recognized by three of these mAbs were the same as those in renatured BLG. However, it is notable that the binding properties of other two mAbs to native BLG indicated a wide structural difference in the epitope regions between the native and renatured BLG. These regions unable to completely refold were the same as those that unfolded preferentially to the alpha-helix region, shown in the previous report (S. Kaminogawa et al., (1989) Biochim. Biophys. Acta 998, 50-56). Complete refolding was never attained by several renaturation conditions such as quicker or slower removal of the denaturant, nor by additional oxidation treatment after reducing the disulfide bonds. These results suggest that some specific moiety(s) in a protein molecule cannot return to the native conformation from a denatured state, even if the other moieties refold completely, and that such a conformational difference between renatured and native forms does not affect on biological function of ligand binding.