Fluorescent Phosphonate Labels for Serine Hydrolases

摘要

This paper reports the synthesis, kinetics of inhibition and reactivation, and absorption, fluorescence and circular dichroism spectra of a homologous pair of methylphosphonofluoridates containing aminoalkyl conjugates of NBD-C1. The new probes are designated NBD-aminoethyl and NBD-aminopentyl methylphosphonofluoridate, NBD-AE-MPF and NBD-AP-MPF, respectively, and by virtue of the different chain lengths separating the fluorophore from the reactive methylphosphonyl group, their spectroscopic characteristics serve to report on the protein environment within different regions of the active center. These probes are employed to examine acetylcholinesterase from Torpedo californica. When isolated by tryptic digestion of Torpedo electroplax, the enzyme exists as a tetrameric species exhibiting a sedimentation coefficient of 11 S (9). Low salt extraction in the absence of tryptic digestion affords, in contrast, a dimeric species characterized by a sedimentation coefficient of 5.6 S (10, 11). Both NBD-AE-MPF and NBD-AP-MPF are found to be potent and specific inhibitors of the 11 S lytic form of acetylcholinesterase and form conjugates with a stoichiometry of one methylphosphonyl moiety/subunit. The spectral characteristics of these homologous probes conjugated with the different molecular forms of acetylcholinesterase are presented and compared with those determined for the corresponding conjugates with alpha-chymotrypsin.