Purification of horse serum cholinesterase by preparative polyacrylamide gel electrophoresis.

Preparative polyacrylamide gel electrophoresis has been used in the final stages of purification to prepare horse serum cholinesterase which was at least 9.5% pure, as judged by analytical polyacrylamide gel electrophoresis. The starting material, already purified about 109-fold by the Strelitz method, was purified an additional 89-fold by the procedure. The method involved Sephadex G-ZOO and Sepharose 6B gel filtration followed by two successive preparative polyacrylamide gel electrophoresis steps. The main problem involved a polyacrylamide gel contaminant. The molecular weight of cholinesterase by Sepharose 6B gel filtration was 315,000. The weight per active site of 143,500 was estimated from diisopropyl phosphorofluoridate titrations and dry weight determinations. The subunit weight of 77,300 was determined by sodium dodecyl sulfate analytical polyacrylamide gel electrophoresis. A cholinesterase molecule consisting of two active sites and four subunits was indicated. Purified preparations were stable to dialysis, high dilutions, and during prolonged storage. The kinetic behavior of the purified material was complex, suggesting more than one form. With butyrylcholine a K, of 0.29 mM and a turnover number of 171,000 min-’ was obtained at 25”, pH 7.6. A K, of 0.53 mM and a turnover number of 61,020 min-l was obtained with acetylthiocholine at 25”, pH 8.1.