UNFOLDING REFOLDING STUDIES ON BOVINE BETA-LACTOGLOBULIN WITH MONOCLONAL-ANTIBODIES AS PROBES - DOES A RENATURED PROTEIN COMPLETELY REFOLD

We investigated whether any local moieties within a protein molecule c ould completely refold from the denatured state to regain the native c onformation. Bovine beta-lactoglobulin (beta-LG) was denatured in the presence of guanidine hydrochloride (GdnHCl) as the denaturant. Renatu ration of the denatured beta-LG was attempted by dialyzing to remove G dnHCl. The renatured molecules regained the same retinol binding activ ity as that of native beta-LG, and physicochemical studies also indica ted that refolding of the denatured beta-LG had been almost completely successful. Local structural differences between the native and renat ured beta-LG molecules were evaluated by using our panel of four anti- beta-LG monoclonal antibodies (anti-beta-LG mAbs). The structures of t he epitope regions in native beta-LG recognized by two of these mAbs w ere the same as those in renatured beta-LG. However, it is notable tha t the binding properties of the other two mAbs to native beta-LG indic ated a wide structural difference in the epitope regions between the n ative and renatured beta-LG. These regions unable to completely refold were the same as those that unfolded preferentially to the alpha-heli x region, shown in the previous report (Kaminogawa, S., Shimizu, M., A metani, A., Hattori, M., Ando, O., Hachimura, S., Nakamura, Y., Totsuk a, M., and Yamauchi, K. (1989) Biochim. Biophys. Acta 998, 50-56). Com plete 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(ies) in a protein molecule cannot r eturn to the native conformation from a denatured state, even if the o ther moieties refold completely, and that such a conformational differ ence between renatured and native forms has no affect on the biologica l function of ligand binding.