Immobilized liposome chromatography for studies of protein-membrane interactions and refolding of denatured bovine carbonic anhydrase.
Small unilamellar vesicles (SUVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1 mol% phosphatidylethanolamine were covalently coupled to chromatographic gel beads. Interactions of liposomal lipid bilayers with several water-soluble proteins, which had been denatured or partially denatured by 0.1-5 M guanidinium hydrochloride (GuHCl), were studied on gel beads containing the immobilized SUVs. The partially-denatured proteins treated with 0.5-1.0 M GuHCl were significantly retarded on the immobilized liposome column, whereas little retardation of native or unfolded proteins treated by >2 M GuHCl was observed on the same liposome columns. The retardation on the immobilized liposome column was found to be well correlated with local hydrophobicity, which was determined by the aqueous two-phase partitioning method using 1 mM Triton X-405 as a hydrophobic probe. It implies that the partially-denatured proteins are likely in a molten-globule state and associated with liposomal lipid bilayers. Chromatographic refolding of denatured bovine carbonic anhydrase (CAB) was achieved on the immobilized liposome column. The enzymatic activity of an unfolded CAB treated by 5 M GuHCl was recovered up to 83% after passing it through immobilized liposome column, whereas only 58% of the enzymatic activity was recovered when the denatured CAB was run on a liposome-free column. The refolding process is probably involved in the interaction of molten-globule state of CAB with the liposomal lipid bilayers.
Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Japan.
M
M
Yoshimoto
R
R
Kuboi
Q
Q
Yang
J
J
Miyake
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005782:Gels; D008027:Light; D008081:Liposomes / Q000737:chemistry; D008863:Microspheres; D010316:Particle Size; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D013499:Surface Properties
D005782:Gels; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D011506:Proteins; C034973:1-oleoyl-2-palmitoylphosphatidylcholine; D002256:Carbonic Anhydrases