Human-Serum Cholinesterase Subunits and Number of Active Sites of the Major Component

Abstract

The major C₄ component of human serum cholinesterase was highly purified by a two-step procedure involving chromatography on DEAE-cellulose and preparative disc electrophoresis. The final product was about 8000-fold purified with a yield of 64%. The subunit structure was determined by 8M urea polyacrylamide disc electrophoresis and by the sedimentation equilibrium centrifugation method in 5M guanidine hydrochloride. It was found that the C₄ enzyme has a tetrameric structure. The subunits are equal in size and charge and a molecular weight comparable to that of the C₁ enzyme from native serum. The major C₄ enzyme and the minor C₁ enzyme were subjected to an ‘active enzyme centrifugation’. It was found that the C₄ enzyme was a tetramer and the C₁, enzyme was a monomer in the presence of substrate. The number of diisopropylphosphofluoridate-binding sites was measured from the molar ratio of bound diisopropylphosphate to protein. A value close to two binding sites was found for the C₄ enzyme.