FLUORESCENCE AND N.M.R. STUDIES OF THE BINDING OF CHOLINERGIC FLUORESCENT PROBES TO HORSE SERUM CHOLINESTERASE

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Abstract
The interaction of the cholinergic fluorescent probes, 1-(5-dimethylaminoaphthalene-1-sulfonamido) ethane-2-trimethylammonium perchlorate, 1-(5-dimethylaminonaphthalene-1-sulfonamido) pentane-5-trimethylammonium tartarate and 1-(5-dimethylaminonaphthalene-1-sulfonamido) decane-10- trimethylammonium tartarate with horse serum cholinesterase was examined by fluorescence and NMR methods. Fluorescence titrations show binding of the decane derivative to 2 sites on the protein, while the lower homologs bind largely to 1 site. Active site inhibitors like carbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. NMR studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative; in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being 2 orders of magnitude more effective than its lower homologs. Based on the NMR, fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, NMR and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.