Molecular Pharmacology, Vol 12, 667-677, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

Interaction of Spin-Labeled Bisquaternary Ammonium Ligands with Acetylcholinesterase

¹ Section on Molecular Pharmacology, Pulmonary Branch, National Heart and Lung Institute, Bethesda, Maryland 20014
² Division of Pharmacology, Department of Medicine, University of California, San Diego, La Jolla, California 92037

The interaction of a mononitroxide analogue of decamethonium (I) and symmetrical dinitroxide analogues of decamethonium (II) and hexamethonium (III) with Torpedo californica acetylcholinesterase has been studied by means of electron spin resonance spectroscopy. The I₅₀ values for inhibition of acetylcholinesterase by labels I-III were 0.25 µM, 0.18 µM, and 3.3 µM, respectively. The ESR spectrum of I bound to acetylcholinesterase indicated that the nitroxide group was highly immobilized. Methanesulfonation of the active site serine of acetylcholinesterase resulted in a 6-fold decrease in the affinity of the enzyme for label I. This suggests that, in the unmodified enzyme, label I probably binds with the nitroxide group at the active site. In aqueous solution, at temperatures above 50°, the ESR spectrum of label II showed evidence for intramolecular electron spin-spin exchange, which was abolished by dissolving the label in ethylene glycol and cooling to -30°. When II bound to acetylcholinesterase, both nitroxide groups of the label became highly immobilized and the spin-spin exchange interaction was abolished. These findings indicate that label II binds to acetylcholinesterase in an extended conformation via a 2-point attachment involving both quaternary nitrogens. The observation that the ESR spectra of labels I and II bound to acetylcholinesterase were independent of fractional site occupation suggests that the bisquaternary binding sites are removed from the axis or center of symmetry of this tetrameric enzyme.