Evaluation of surface hydrophobicities during refolding process of carbonic anhydrase using aqueous two-phase partitioning systems.

Abstract

Changes in conformational and surface properties in refolding processes were quantitatively investigated in aqueous two-phase systems, using bovine carbonic anhydrase (CAB) as a model protein. Surface net hydrophobicity (HFS) of a native CAB was determined as –84 kj·mol–1, showing a moderately hydrophilic surface. By addition of 1.5–2 M guanidine hydrochloride (GuHCl), CAB was denatured and HFS increased drastically up to about 300 kj·mol–1, accompanied by some increment of local hydrophobicity. The fully unfolded state was achieved in 5 M GuHCl. The unsteady change of local hydrophobicity during CAB refolding was also quantified using phase separation of Triton solution. Yield of CAB reactivation was enhanced by the addition of Triton X-405, due to hydrophobic interaction with hydrophobic residues exposed during the refolding process.