Stability, inhibition and reactivation of acetylcholinesterase covalently coupled to glass

Abstract

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was covalently coupled to a silanized fritted glass disc by a diazonium salt reaction and by a diimide condensation. The diimide-coupled adduct survived numerous assays and chemical studies for a period of 55 days at room temperature in contrast to the diazonium-coupled adduct which rapidly lost activity. Both adducts exhibited a lesser pronounced dependence of activity on pH than the free enzyme, reflecting the weak buffering action of the silanol-silane surface. The diimide adduct is inhibited by diethyl p-nitrophenyl phosphate and can be reactivated by either Toxigonin or alkaline phosphatase (orthophosphoric monoester phosphohydralase, EC 3.1.3.1).