Oxidative refolding of lysozyme assisted by negatively charged liposomes: relationship with lysozyme-mediated fusion of liposomes.
Oxidative refolding of denatured/reduced lysozyme was examined in the presence of charged liposomes composed of neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Surface charge density of liposomes had a marked effect on the interaction between liposomes and reduced lysozyme which is observed in the early stage of the refolding. It was found that there was a critical level of surface charge density of liposomes (-0.15 C/nm2) at which the interaction between liposomes and lysozyme drastically changed. At the surface charge density of liposomes ranging from -0.15 to -1.4 C/nm2, the interaction between liposomes and lysozyme resulted in aggregate formation. In contrast, at the surface charge density ranging from 0 to -0.15 C/nm2, no aggregate formation was observed if the lysozyme/liposome molar ratio was less than 600. On the basis of the experimental results, a model for the interaction between charged liposomes and lysozyme was proposed, focusing on the mechanism of protein-induced fusion of charged liposomes as well as protein refolding on liposomes. Then, the optimal condition for oxidative refolding of lysozyme was examined in the presence of charged liposomes by controlling the lysozyme-liposome interaction. The reactivation yield of lysozyme was improved up to 85% in the presence of liposomes with a surface charge density of -0.14 C/nm2.
Kuboi
R
R
Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan.
Mawatari
T
T
Yoshimoto
M
M