Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy.
Abstract 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 substrate…
In vitro and in vivo efficacy of PEGylated diisopropyl fluorophosphatase (DFPase)
Highly toxic organophosphorus compounds that irreversibly inhibit the enzyme acetycholinesterase (AChE), including nerve agents like tabun, sarin, or soman, still pose a credible threat to civilian populations and military personnel. New therapeutics that can be used as a pretreatment or after poisoning with these compounds, complementing existing treatment schemes such as the use of atropine and AChE reactivating oximes, are currently the subject of intense research. A prominent role among potential candidates is taken by enzymes that can detoxify nerve agents by hydrolysis. Diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is known to effectively hydrolyze DFP and the …
Reversed Enantioselectivity of Diisopropyl Fluorophosphatase against Organophosphorus Nerve Agents by Rational Design
Diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is an efficient and robust biocatalyst for the hydrolysis of a range of highly toxic organophosphorus compounds including the nerve agents sarin, soman, and cyclosarin. In contrast to the substrate diisopropyl fluorophosphate (DFP) the nerve agents possess an asymmetric phosphorus atom, which leads to pairs of enantiomers that display markedly different toxicities. Wild-type DFPase prefers the less toxic stereoisomers of the substrates which leads to slower detoxification despite rapid hydrolysis. Enzyme engineering efforts based on rational design yielded two quadruple enzyme mutants with reversed enantioselectivity and overall en…
Stable adducts of nerve agents sarin, soman and cyclosarin with TRIS, TES and related buffer compounds—Characterization by LC-ESI-MS/MS and NMR and implications for analytical chemistry
Abstract Buffering compounds like TRIS are frequently used in chemical, biochemical and biomedical applications to control pH in solution. One of the prerequisites of a buffer compound, in addition to sufficient buffering capacity and pH stability over time, is its non-reactivity with other constituents of the solution. This is especially important in the field of analytical chemistry where analytes are to be determined quantitatively. Investigating the enzymatic hydrolysis of G-type nerve agents sarin, soman and cyclosarin in buffered solution we have identified stable buffer adducts of TRIS, TES and other buffer compounds with the nerve agents. We identified the molecular structure of the…