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Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of diisopropyl fluorophosphatase (DFPase) with 1H–31P HSQC NMR spectroscopy

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Abstract

The enzyme diisopropyl fluorophosphatase (DFPase, EC 3.1.8.2) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Until now, determination of kinetic data has been achieved by use of techniques such as pH-stat titration, ion-selective electrodes, and a recently introduced method based on in situ Fourier-transform infrared (FTIR) spectroscopy. We report the use of 1D 1H–31P HSQC NMR spectroscopy as a new method for real-time quantification of the hydrolysis of toxic organophosphonates by DFPase. The method is demonstrated for the agents sarin (GB), soman (GD), and cyclosarin (GD) but can also be used for V-type nerve agents, for example VX. Besides buffered aqueous solutions the method was used to determine enzymatic activities in a biodiesel-based bicontinuous microemulsion that serves as an example of complex decontamination media, for which other established techniques often fail. The method is non-invasive and requires only limited manual handling of small volumes of liquid (700 μL), which adds to work safety when handling highly toxic organophosphorus compounds. Limits of detection are slightly below 100 μmol L−1 on a 400 MHz spectrometer with 16 FIDs added for a single time frame. The method is not restricted to DFPase but can be used with other phosphotriesterases, for example paraxonase (PON), and even reactive chemicals, for example oximes and other nucleophiles, as long as the reaction components are compatible with the NMR experiment.

Staggered view of consecutive 1D 1H–31P HSQC NMR spectra recorded during the hydrolysis of GF catalyzed by DFPase in aqueous buffer solution. During the course of the enzyme-catalyzed reaction the doublet of doublets (dd) signal of the nerve agent is decreasing and at the same time the doublet (d) signal of the phosphonate is increasing. The phosphonate (d) signal is used for quantification because of a better signal-to-noise ratio compared with the (dd) of the agent

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References

  1. Convention on the prohibition of the development, production, stockpiling and use of chemical weapons and on their destruction; Technical Secretariat of the Organisation for the Prohibition of Chemical Weapons: The Hague, 1997

  2. Vale A (2005) What lessons can we learn from the Japanese sarin attacks? Przegl Lek 62:528–532

    Google Scholar 

  3. Scharff EI, Koepke J, Fritzsch G, Lücke C, Rüterjans H (2001) Crystal structure of diisopropylfluorophosphatase from Loligo vulgaris. Structure 9:493–502

    Article  CAS  Google Scholar 

  4. Blum MM, Löhr F, Richardt A, Rüterjans H, Chen JCH (2006) Binding of a designed substrate analogue to diisopropyl fluorophosphatase: implications for the phosphotriesterase mechanism. J Am Chem Soc 128:12750–12757

    Article  CAS  Google Scholar 

  5. Blum MM, Mustyakimov M, Rüterjans H, Kehe K, Schönborn BP, Langan P, Chen JCH (2009) Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement. Proc Natl Acad Sci USA 106:713–718

    Article  CAS  Google Scholar 

  6. Gäb J, Melzer M, Kehe K, Richardt A, Blum MM (2009) Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy. Anal Biochem 385:187–193

    Article  Google Scholar 

  7. Hartleib J, Rüterjans H (2001) High-yield expression, purification, and characterization of the recombinant diisopropylfluorophosphatase from Loligo vulgaris. Protein Expr Purif 21:210–219

    Article  CAS  Google Scholar 

  8. Hoskin FCG, Long RJ (1972) Purification of a DFP- hydrolyzing enzyme from squid head ganglion. Arch Biochem Biophys 150:548–555

    Article  CAS  Google Scholar 

  9. Hoskin FCG, Roush AH (1982) Hydrolysis of nerve gas by squid-type diisopropyl phosphorofluoridate hydrolyzing enzyme on agarose resin. Science 215:1255–1257

    Article  CAS  Google Scholar 

  10. Hartleib J, Rüterjans H (2001) Insights into the reaction mechanism of the diisopropyl fluorophosphatase from Loligo vulgaris by means of kinetic studies, chemical modification and site-directed mutagenesis. Biochim Biophys Acta 1546:312–324

    CAS  Google Scholar 

  11. Amitai G, Gaidukov L, Adani R, Yishay S, Yacov G, Kushnir M, Teitlboim S, Lindenbaum M, Bel P, Khersonsky O, Tawfik DS, Meshulam H (2006) Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase. FEBS J 273:1906–1919

    Article  CAS  Google Scholar 

  12. Briseno-Roa L, Hill J, Notman S, Sellers D, Smith AP, Timperley CM, Wetherell J, Williams NH, Williams GR, Fersht AR, Griffiths AD (2006) Analogues with fluorescent leaving groups for screening and selection of enzymes that efficiently hydrolyze organophosphorus nerve agents. J Med Chem 49:246–255

    Article  CAS  Google Scholar 

  13. Blum MM, Timperley CM, Williams GR, Thiermann H, Worek F (2008) Inhibitory potency against human acetylcholinesterase and enzymatic hydrolysis of fluorogenic nerve agent mimics by human paraoxonase 1 and squid diisopropyl fluorophosphatase. Biochemistry 47:5216–5224

    Article  CAS  Google Scholar 

  14. Wellert S, Karg M, Imhof H, Steppin A, Altmann HJ, Dolle M, Richardt A, Tiersch B, Koetz J, Lapp A, Hellweg T (2008) Structure of biodiesel based bicontinuous microemulsions for environmentally compatible decontamination: A small angle neutron scattering and freeze fracture electron microscopy study. J Colloid Interface Sci 325:250–258

    Article  CAS  Google Scholar 

  15. Hellweg T, Wellert S, Mitchell SJ, Richardt A (2008) Microemulsions: a versatile carrier for decontamination agents. In: Richardt A, Blum MM (eds) Decontamination of warfare agents – Enzymatic methods for the removal of B/C weapons. Wiley–VCH, Weinheim

    Google Scholar 

  16. Mesilaakso M, Tolppa EL (1996) Detection of trace amounts of chemical warfare agents and related compounds in rubber, paint and soil samples by 1H and 31P{1H} NMR spectroscopy. Anal Chem 68:2313–2318

    Article  CAS  Google Scholar 

  17. Albaret C, Loeillet D, Augé P, Fortier PL (1997) Application of two-dimensional 1H–31P inverse NMR spectroscopy to the detection of trace amounts of organophosphorus compounds related to the chemical weapons convention. Anal Chem 69:2694–2700

    Article  CAS  Google Scholar 

  18. Meier UC (2004) Application of nonselective 1D 1H–31P inverse NMR spectroscopy to the screening of solutions for the presence of organophosphorus compounds related to the chemical weapons convention. Anal Chem 76:392–398

    Article  CAS  Google Scholar 

  19. Koskela H, Rapinoja ML, Kuitunen ML, Vanninen P (2007) Determination of trace amounts of chemical warfare agent degradation products in decontamination solutions with NMR spectroscopy. Anal Chem 79:9098–9106

    Article  CAS  Google Scholar 

  20. Raushel FM (2002) Bacterial detoxification of organophosphate nerve agents. Curr Opin Microbiol 5:288–295

    Article  CAS  Google Scholar 

  21. Cheng TC, DeFrank JJ, Rastogi VK (1999) Alteromonas prolidase for organophosphorus G-agent decontamination. Chem Biol Interact 119–120:455–462

    Article  Google Scholar 

  22. Lenz DE, Yeung D, Smith JR, Sweeney RE, Lumley LA, Cerasoli DM (2007) Stoichiometric and catalytic scavengers as protection against nerve agent toxicity: a mini review. Toxicology 233:31–39

    Article  CAS  Google Scholar 

  23. Deutsche Forschungsgemeinschaft (1991) Rückstandsanalytik von Pflanzenschutzmitteln, 11:XI-A-1. VCH, Weinheim

    Google Scholar 

  24. Frehse H, Their HP (1991) The limits of detection and of determination in residue analyses – their derivation according to the new DFP-approach. GIT Fachz Lab 35:285–291

    CAS  Google Scholar 

  25. Burton IW, Quilliam MA, Walter JA (2005) Quantitative 1H NMR with external standards: use in preparation of calibration solutions for algal toxins and other natural products. Anal Chem 77:3123–3131

    Article  CAS  Google Scholar 

  26. Quinn LD (2000) Organophosphorus chemistry. Wiley–Interscience, New York, pp 169–172

    Google Scholar 

Download references

Acknowledgements

The German Ministry of Defense supported this work under contract number E/UR3G/6G115/6A801. The authors thank Dr Harri Koskela (Verifin, University of Helsinki, Finland) for kindly providing pulse sequence files for 1H–31P HSQC NMR experiments and Professor Julian C.-H. Chen (Institute of Biophysical Chemistry, University of Frankfurt, Germany) for his support and productive discussions.

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Authors and Affiliations

  1. Blum – Scientific Services, Ledererstrasse 23, 80331, Munich, Germany

    Jürgen Gäb, Marco Melzer & Marc-Michael Blum

  2. Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032, Marburg, Germany

    Jürgen Gäb

  3. Institute of Pathology, Johannes Gutenberg University Mainz, Langenbeckstrasse 1, 55101, Mainz, Germany

    Marco Melzer

  4. Bundeswehr Institute for Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany

    Kai Kehe

  5. Helmholtz Zentrum Berlin für Materialien und Energie, Glienicker Strasse 100, 14109, Berlin, Germany

    Stefan Wellert

  6. Physical Chemistry I, University of Bayreuth, 95440, Bayreuth, Germany

    Thomas Hellweg

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  1. Jürgen Gäb
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  2. Marco Melzer
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  3. Kai Kehe
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  5. Thomas Hellweg
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  6. Marc-Michael Blum
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Correspondence to Marc-Michael Blum.

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Gäb, J., Melzer, M., Kehe, K. et al. Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of diisopropyl fluorophosphatase (DFPase) with 1H–31P HSQC NMR spectroscopy. Anal Bioanal Chem 396, 1213–1221 (2010). https://doi.org/10.1007/s00216-009-3299-2

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  • DOI: https://doi.org/10.1007/s00216-009-3299-2

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