Structure of human erythrocyte acetylcholinesterase. Characterization of intersubunit disulfide bonding and detergent interaction

A large scale purification procedure for human erythrocyte acetylcholinesterase that involved affinity chromatography on an acridinium resin permitted the routine isolation of about 5 mg of enzyme from 10 liters of outdated erythrocytes. The purified enzyme had a specific activity of 5000-5800 units/mg of protein and was free of polypeptide contaminants by gel electrophoresis criteria. In detergents, the isolated enzyme corresponded to a disulfide-linked dimer (G₂) that was converted to 75-kDa subunit monomers (G₁) by reduction with dithiothreitol. No free sulfhydryl groups were detected prior to reduction, but reduction under nondenaturing conditions generated active G₁ and produced 1.7 mol of free sulfhydryl groups/mol of subunit. These data were interpreted as indicating a single intersubunit disulfide bond in the G₂ enzyme. In the absence of nonionic detergents, both the G₂ and the G₁ enzymes formed aggregates with average Stokes radii of 10 nm. Introduction of Triton X-100 gave enzyme-detergent complexes according to hydrodynamic criteria. Quantitative determination of [³H]Triton X-100 binding to G₂ and G₁ by a novel affinity chromatography procedure revealed that each G₂ molecule bound about 140 detergent molecules and each G₁, about 80. These observations indicated that each subunit in both G₂ and G₁ interacted individually with a Triton X-100 micelle. Molecular weight estimates for the protein components of the G₂- and G₁-detergent complexes were obtained from the hydrodynamic properties and the detergent binding data and corresponded to 160,000 and 85,000, respectively. Data in this and the accompanying paper (Dutta-Choudhury, T.A., and Rosenberry, T.L. (1984) J. Biol. Chem. 259, 5653-5660) provide strong evidence that erythrocyte acetylcholinesterase is an amphipathic protein.