Degradation of nerve gases by CLECS and cells: kinetics of heterogenous systems.
We have reported the enzymatic hydrolysis of phosphoro- and phosphonofluoridates and phosphoro- and phosphonothiolates and -thionates by an organophosphorus hydrolase (OPH) from Pseudomonas diminuta. In screening for other microbial sources of nerve gas hydrolyzing enzymes, it would be convenient, indeed essential, to be able to determine such hydrolyses on intact cells. As a preliminary step to such screening we have measured the hydrolysis of O,O-diisopropyl S-(2-diisopropylaminoethyl) phosphorothiolate (Tetriso) and O,O-diethyl S-(2-ethylthioethyl) phosphorothiolate (Demeton-S; formerly Isosystox) by intact cells and sonicates. The purified OPH has also been cross-linked to itself (CLEC = cross-linked enzyme crystals) and this has also been tested for its ability to hydrolyze Tetriso and Demeton-S. The testing of such heterogenous systems by a spectrophotometric assay (Ellman) has required novel modifications. Our findings are that both Tetriso and Demeton-S are subject to intact-cell assay, that both are readily hydrolyzed by the CLEC-ed OPH without marked change in kinetics, but that at any given substrate concentration Tetriso is hydrolyzed much more rapidly. However, since Demeton-S is commercially available, this appears to be the substrate most suitable for screening for our final goal in a search for sources of enzymes to detoxify O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX).
Marine Biological Laboratory, Woods Hole, MA 02543, USA.
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Hoskin
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Walker
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Stote
D043303:Aryldialkylphosphatase; D001673:Biodegradation, Environmental; D002619:Chemical Warfare Agents / Q000378:metabolism / Q000493:pharmacokinetics*; D003432:Cross-Linking Reagents; D003460:Crystallization; D004950:Esterases / Q000378:metabolism*; D006868:Hydrolysis; D008658:Inactivation, Metabolic; D007306:Insecticides / Q000378:metabolism / Q000493:pharmacokinetics; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000378:metabolism / Q000493:pharmacokinetics*; D063086:Organothiophosphates / Q000378:metabolism / Q000493:pharmacokinetics*; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry
D002619:Chemical Warfare Agents; D003432:Cross-Linking Reagents; D007306:Insecticides; D009943:Organophosphorus Compounds; D063086:Organothiophosphates; C091319:Tetriso; D004950:Esterases; D043303:Aryldialkylphosphatase; C030064:demeton-S-methyl