Multiparameter kinetic study on the unfolding and refolding of bovine carbonic anhydrase B.

The kinetics of unfolding and refolding of bovine carbonic anhydrase B by guanidinium chloride have been studied by simultaneously monitoring several spectroscopic parameters, each of which reflects certain unique conformational features of the protein molecule. In the present report, far-UV circular dichroism (CD) was used to follow the secondary structural change, UV difference absorption was used to follow the exposure or burying of aromatic amino acid residues, and near-UV CD was used to follow tertiary structural changes during unfolding and refolding. The unfolding is described by two unimolecular rate processes, and refolding is described by three unimolecular rate processes. The minimum number of conformational species involved in the mechanism is five. The refolding of the protein followed by the above three parameters indicates that the process consists of an initial rapid phase in which the random-coiled protein is converted to an intermediate state(s) having secondary structure comparable to that of the native protein. This is followed by the burying of the aromatic amino acid residues to form the interior of the protein molecule. Subsequently, the protein molecule acquires its tertiary structure and folds into a unique conformation with the formation of aromatic clusters.