Isolation of human erythrocyte acetylcholinesterase using phase separation with Triton X-114 and monoclonal immunosorbent chromatography.
A generally applicable approach to the preparative isolation of amphiphilic membrane proteins that follow the Triton X-114 phase during a temperature-dependent phase separation is described. The phase separations were performed direct on whole blood and a 650-fold purification of human erythrocyte membrane acetylcholinesterase (AchE) was obtained. Thus, 0.2 mg enzyme was isolated per 1 liter of blood, with a specific activity of 13 IU/mg, the major contaminants being glycophorin and hemoglobin. The protein material was isolated from the detergent phase by Cu2+ chelate chromatography. This material was used to raise monoclonal anti-AchE antibodies which, when applied to immunosorbent chromatography of washed Triton X-100-lysed erythrocytes in one step, allowed a 246,000-fold purification of AchE with a yield of 88% and a specific activity of 3800 IU/mg.
D000110:Acetylcholinesterase / Q000097:blood*; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D004910:Erythrocyte Membrane / Q000201:enzymology; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007163:Immunosorbent Techniques; D066298:In Vitro Techniques; D017830:Octoxynol; D011092:Polyethylene Glycols
D000911:Antibodies, Monoclonal; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D000110:Acetylcholinesterase