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.
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