Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy

被引:28
作者
Gaeb, Juergen [1 ,2 ]
Melzer, Marco [1 ,3 ]
Kai Kehe [4 ]
Richardt, Andre [5 ]
Blum, Marc-Michael [1 ]
机构
[1] Blum Sci Serv, D-80331 Munich, Germany
[2] Univ Marburg, Inst Pharmaceut Chem, D-35032 Marburg, Germany
[3] Johannes Gutenberg Univ Mainz, Inst Pathol, D-55101 Mainz, Germany
[4] Bundeswehr Inst Pharmacol & Toxicol, D-80937 Munich, Germany
[5] Armed Forces Sci Inst Protect Technol NBC Protect, D-29633 Munster, Germany
关键词
Phosphotriesterases; DFPase; Nerve agents; DFP; FTIR; Infrared spectroscopy; Enzymatic assay; Hydrolases; ENZYMATIC-ACTIVITY; ASSAY; DIISOPROPYLFLUOROPHOSPHATASE; PURIFICATION;
D O I
10.1016/j.ab.2008.11.012
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The enzyme diisopropyl fluorophosphatase (DFPase) 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. Up to now, the determination of kinetic data has been achieved by techniques Such as pH-stat titration, ion-selective electrodes, and fluorogenic substrate analogs. We report a new assaying method using in situ Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR) for the real-time determination of reaction rates. The method employs changes in the P-O-R stretching vibration of DFP and nerve agent substrates when hydrolyzed to their corresponding phosphoric and phosphonic acids. It is shown that the Limbert-Beer law holds and that changes in absorbance can be directly related to changes in concentration. Compared with other methods, the use of in situ FTIR spectroscopy results in a substantially reduced reaction volume that adds extra work safety when handling highly toxic substrates. In addition, the new method allows the noninvasive measurement of buffered solutions with varying ionic strengths complementing existing methods. Because the assay is independent of the used enzyme, it should also be applicable to other phosphotriesterase enzymes such as organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA), and paraoxonase (PON). (c) 2008 Elsevier Inc. All rights reserved.
引用
收藏
页码:187 / 193
页数:7
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