Covalent Affinity Chromatography of Acetylcholinesterase

Abstract

Active site directed inhibitors of the active serine residue of the catalytic center of acetylcholinesterase can be immobilized by covalently bonding them to derivatized agarose gels. If the immobilized inhibitor is an “irreversible” inhibitor, then not only is there a high degree of specificity for acetylcholinesterase, but there is also a certain amount of stability achieved because the enzyme will be covalently bonded to the gel. These considerations prompted Ashani and Wilson (1) to design an affinity gel that had as its ligand a methylphosphonate derivative which is an active site directed irreversible inhibitor of acetylcholinesterase. Eq. 1 demonstrates the general features of the binding,where E is acetylcholinesterase and the -OH of E-OH is the hydroxyl group of the active serine at the esteratic site; the inhibitor is aminoethyl p-nitrophenylmethylphosphonate, R is succinyldiaminopentylagarose gel, and p-nitrophenol is the leaving group. It has been demonstrated that this immobilized methylphosphonate has good specificity for acetylcholinesterase from electric organs ofElectrophorus eZectricus,Torpedo californica, as well as for α-chymotrypsin (1, 4–7).