[Inhibition of acetylcholinesterase using fluorescence-labeled inhibitors].

The inhibition of acetylcholinesterase with fluorophores type II and III linked with the OH-selective fluorophosphonoyl groups is kinetically investigated by comparing the changes in activity and fluorescence. The hydrolysis of the fluorescent phosphonoylfluorides 1 and 4 to 7 in aqueous buffer solutions does not interfere with the inhibition kinetics. The inhibition constants of the investigated compounds are unexpectedly high (10(6) to 10(8) S-1M-1). They increase with increasing spacer length, but arrive at an optimal value with four methylene groups in the inhibitor 6. The fluorescence is quenched by the interaction of 6 and acetylcholinesterase. This fact can be used for the determination of acetylcholinesterase by fluorescence titration (Fig. 9). Fluorescence once more increases slowly during the aging process, leading to the degradation products 9, 11 and 12. In acetylcholinesterase inhibited by 1, a sensitized fluorescence is observed, produced by tryptophane intrinsically linked to the esterase. In the presence of quaternary ammonium salts like acetylcholine chloride or gallamine triethochloride 15, the decrease of fluorescence is lower. Acetylcholinesterase inhibited in this way is reactivated quantitatively by toxogonine. No reactivation is possible with acetylcholinesterase inhibited in the absence of the above mentioned quaternary ammonium salts. As a result of the investigation using fluorescent inhibitors the conclusion can be drawn that not only the active site of acetylcholinesterase is blocked by phosphonoylation. The conformation too seems to be influenced by interactions of the inhibitors with the hydrophobic areas of the enzyme.