Propidium (3, 8-diamino-5, 3'-diethylmethylamino-n-propyl-6-phenylphenanthridium) diiodide binds with high affinity to a purified acetylcholinesterase isolated by lytic procedures from Torpedo californica. Complex formation results in 10-fold enhancement of fluorescence for the ligand in the bound state. The fluorescent ligand can be dissociated from the enzyme by back-titration with d-tubocurarine and gallamine under conditions of low ionic strength (Γ/2 ≅ 0.001). At higher ionic strength, in 0.1 M NaCl, 0.04 M MgCl2, and 0.01 M Tris-Cl, pH 8.0 (Γ/2 = 0.23), the above ligands are relatively ineffective in dissociating propidium from the enzyme. Edrophonium, an inhibitor that appears specific for the active center, does not dissociate propidium under conditions of high and low ionic strength. This specificity is consistent with propidium acting as a fluorescence probe for a peripheral anionic site on acetylcholinesterase.
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