Choline oxidase: a useful tool for high-throughput assays of acetylcholinesterase, phospholipase D, phosphatidylcholine-specific phospholipase C, and sphingomyelinase

Choline oxidase converts one molecule of choline to generate two molecules of H2O2. In a 1:1 reaction stoichiometry, horseradish peroxidase-catalyzed oxidation of the nonfluorescent and colorless H2O2 indicator, the Amplex Red reagent, by H2O2 produces the highly fluorescent resorufin, which has an excitation maximum at 563 nm and emission maximum at 587 nm. Using the Amplex Red reagent as an H2O2 indicator, assays of phospholipase D, phosphatidylcholine-specific phospholipase C, acetylcholine, acetylcholinesterase, and sphingomyelinase have been developed in choline oxidase- and HRP-coupled reactions. These assays have been developed in a microplate with a one-step procedure.