FURTHER EVIDENCE ON THE EQUILIBRIUM PRE-MOLTEN GLOBULE STATE - 4-STATE GUANIDINIUM CHLORIDE-INDUCED UNFOLDING OF CARBONIC-ANHYDRASE-B AT LOW-TEMPERATURE

Equilibrium guanidinium chloride-induced unfolding of bovine carbonic anhydrase NE has been investigated by a combination of optical methods with size-exclusion chromatography. It has been shown that, as in the case of staphylococcal beta-lactamase, bovine carbonic anhydrase B un folds at low temperature through two equilibrium intermediates; the mo lten globule and the pre-molten globule states. This pre-molten globul e state has a hydrodynamic volume no more than twofold larger than tha t of the native state, i.e. is relatively compact. It has a pronounced far UV CD spectrum, suggesting the presence of a substantial secondar y structure. It binds 8-anilinonaphthalene-1-sulphonate (though weaker than the molten globule state), which suggests the formation of solve nt-exposed clusters of non-polar groups. Thus, this novel state of pro tein molecules shares a number of properties with the ''burst'' kineti c intermediate of protein folding and can be considered as its equilib rium counterpart. (C) 1996 Academic Press Limited