Inactivation of acetylcholinesterase with a bretylium tosylate photoaffinity probe

Azidobretylium tosylate (ABT), the p-azido analogue of bretylium tosylate, has been synthesized to serve as a photoaffinity probe for bretylium binding sites. Bretylium tosylate has antiarrhythmic action and also interacts with amiloride-sensitive sodium ion transport sites. Acetylcholinesterase was used as a model protein, and both bretylium and ABT are reversible inhibitors of this enzyme. The kinetic inhibition constants (Ki) were determined to be 40 microM for bretylium tosylate and 6 microM for ABT. The azido compound is photochemically labile and apparently irreversibly inactivates the enzyme. The rate was retarded by the addition of bretylium tosylate or 4-oxo-N,N,N-trimethylpentanaminium iodide (OTI). Sephadex G-25 chromatography further demonstrated the irreversible nature of the photoinactivation. Since ABT binds at or near the acetylcholinesterase active site, it may be a useful probe for the characterization of the enzyme active site.