Covalent conjugation of tetrameric bovine liver catalase to liposome membranes for stabilization of the enzyme tertiary and quaternary structures.
Tetrameric bovine liver catalase (BLC) is unstable because of its dissociation into subunits at low enzyme concentrations and the conformational change of the subunits at high temperatures. In this work, for stabilization of BLC, the enzyme was covalently conjugated with liposome membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-glutaryl (NGPE). The NGPE, which was responsible for the BLC/membrane coupling, was altered from 0.05 to 0.2 in its liposomal mole fraction f(G). The catalase-conjugated liposome (CCL) with f(G) of 0.15 showed the maximum number of the conjugated BLC molecules of 28 per liposome. The reactivity of CCLs to H(2)O(2) was as high as that of free BLC at 25 degrees C in Tris-HCl buffer of pH 7.4. Among the CCLs, the catalyst with f(G) of 0.15 was the most stable at 55 degrees C in its enzyme activity in the buffer because the appropriate number of BLC/liposome covalent bonding prevented the dissociation-induced enzyme deactivation. Furthermore, the CCL showed much higher stability at 55 degrees C than the free BLC/enzyme-free liposome mixture and free BLC at the low BLC concentration of 340ng/mL. This was because BLC in the CCL was located in the vicinity of the host membrane regardless of the catalyst concentration, which could induce the effective stabilization effect of the membrane on the enzyme tertiary structure as indicated by the intrinsic tryptophan fluorescence analysis. The results obtained demonstrate the high structural stability of BLC in the CCL system, which was derived from the covalent bonding and interaction between BLC and liposomes.
Yoshimoto
Makoto
M
Department of Applied Molecular Bioscience, Yamaguchi University, Ube, Japan. yosimoto@yamaguchi-u.ac.jp
Sakamoto
Hideyuki
H
Shirakami
Hiroshi
H
D000818:Animals; D002374:Catalase / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D004795:Enzyme Stability; D006358:Hot Temperature; D008081:Liposomes / Q000737:chemistry*; D008099:Liver; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D020836:Protein Structure, Quaternary / Q000187:drug effects*; D017434:Protein Structure, Tertiary / Q000187:drug effects*; D013050:Spectrometry, Fluorescence
D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; C020888:1,2-dielaidoylphosphatidylethanolamine; D002374:Catalase; C028694:1-palmitoyl-2-oleoylphosphatidylcholine