Purification of acetylcholinesterase from house fly brain by affinity chromatography

Abstract

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 ( K i ≈ 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; K i ≈ 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 of 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 306 000 (±11 150) for heavy ( s 20, w = 11.5 S ) form and 143 000 (±4700) for light ( s 20, w = 6.9 S ) form.