REFOLDING OF CARBONIC-ANHYDRASE ASSISTED BY 1-PALMITOYL-2-OLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE LIPOSOMES

The interaction between 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholi ne (POPC) liposome and denatured bovine carbonic anhydrase (CAB) was i nvestigated with the aim of developing an effective liposome-assisted protein refolding process. The Liposome-denatured CAB interaction faci litated the recovery of CAB activity. In the presence of liposomes ([P OPC] = 0.25 mM), 78 +/- 7% of guanidinium hydrochloride (GuHCl)-denatu red CAB was reactivated at 25 degrees C (final concentrations of CAB a nd GuHCl were 3.3 mu M and 0.1 M, respectively), while the refolding y ield reached only 50 +/- 5% in the absence of liposomes. Furthermore, the enzymatic activity of refolded CAB was maintained for at least 1.5 h if the CAB refolding operation was carried out in the presence of l iposomes even at high temperature (similar to 55 degrees C). The data obtained can be interpreted as follows. The first step in this liposom e-assisted CAB refolding is the binding of a refolding intermediate of CAB to the outer surface of liposomes mainly due to hydrophobic inter action. At this stage, the formation of inactive intermolecular aggreg ates of CAB was prevented by the interaction with the Liposomes. Subse quently, the native refolded CAB molecules were released spontaneously from the Liposome-CAB complexes. Ttl the liposome-assisted CAB refold ing process, the reactivation yield of CAB was affected by the physica l properties of the liposome membrane. Surface hydrophobicity and flui dity of liposomes, which were dependent on Liposome size and temperatu re, were found to be key parameters for the improvement of the reactiv ation yield of CAB.