Purification of acetylcholinesterase from house fly brain by affinity chromatography

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from the heads of house flies (Musca domestica L.) was purified by affinity chromatography. The enzyme was adsorbed from the crude extracts on an affinity column containing trimethyl(p-aminophenyl) ammonium chloride hydrochloride (Ki approximately 1.7 - 10(-4) M), covalently linked to Sepharose 4B, then eluted with a solution of a selective reversible inhibitor, 1,5-bis (4-allyl dimethyl ammoniumphenyl)-pentan-3-one dibromide (BW 284C51; Ki approximately 1 - 10(-7) M). The enzyme was purified 1223 times in one step and had a specific activity of 752 units/mg protein. Disc gel electrophoresis in polyacrylamide gel revealed five protein bands, four corresponding to the enzyme activity bands and one devoid of enzyme activity. On the basis of periodic acid-Schiff stain intensity, the slower moving isozyme I and the contaminating band appear to be rich in carbohydrate. The purity of the enzyme estimated by disc gel electrophoresis was 94%. Density gradient centrifugation in sucrose showed two major species each of which ran as a single band on disc gel electrophoresis. The average molecular weights were 306000 (+/- 11 150) for heavy (s20,w = 11.5 S) form and 143000 (+/- 4700) for light (s20,w = 6.9 S) form.