ToxSci Advance Access originally published online on August 13, 2007
Toxicological Sciences 2007 100(1):136-145; doi:10.1093/toxsci/kfm215
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Aging Pathways for Organophosphate-Inhibited Human Butyrylcholinesterase, Including Novel Pathways for Isomalathion, Resolved by Mass Spectrometry
* Eppley Institute and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805
Centre de Recherches du Service de Santé des Armées, Département de Toxicologie-Unité d'Enzymologie, 24 avenue des Maquis du Grésivaudan-BP87, 38702 La Tronche cedex, France
1 To whom correspondence should be addressed at Eppley Institute, University of Nebraska Medical Center, Box 986805, 600 South 42nd Street, Omaha, NE 68198. Fax: (402) 559-4651. E-mail: olockrid{at}unmc.edu.
Received June 27, 2007; accepted July 24, 2007
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Abstract |
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Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser–O–P(O)–(Y)–(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X–R moiety is dealkylated by breaking either the P–X or the X–R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O18 to allow incorporation of 18O, if cleavage was at the P–X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25°C, pH 7.0. However, BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O–C bond cleavage, i.e., the classical X–R scission pathway. In contrast, isomalathion aged through both X–R and P–X pathways; the main aged product resulted from P–S bond cleavage and a minor product resulted from O–C and/or S–C bond cleavage.
Key Words: butyrylcholinesterase; organophosphate; aging; mass spectrometry.