Unfolding of Recombinant Single-chain Insulin in Denaturants Containing Thiol Reagents

Recombinant single-chain insulin (PIP) contains three disulfide bonds. In the presence of denaturants and thiol reagents, the native structure of PIP was disturbed and its native disulfides were shuffled to form a mixture of scrambled isomers which have different degrees of unfolding. In this paper the unfolding degrees of PIP in urea or guanidine hydrochloride containing 0.2 mmol/L 2-mercaptoethanol was analyzed by reverse-phase HPLC and far-UV circular dichroism(CD). The peptide mapping of PIP scrambles demonstrated that PIP had shuffled its native disulfides under the condition we used. Among others, a major non-natural PIP disulfide isomer was purified and its refolding in vitro was investigated. These results show that PIP has only one thermodynamically stable disulfide linkage, and the non-natural disulfide isomers could refold in vitro efficiently to from native PIP. On basis of these, the differences between PIP, IGF-I and insulin on unfolding and refolding were discussed.