Determination of EPN, Its Oxygen Analog, and p-Nitrophenol in Aqueous Suspensions and Enzymic Digests

Abstract

Abstract O-Ethyl O-p-nitrophenyl phenylphosphonothioate (EPN), its oxygen analog O-ethyl O-p-nitrophenyl phenylphosphonate (EPNO), and p-nitrophenol (PNP) are completely extractable from slightly acidic aqueous suspensions or enzymic digests by a mixture of equal volumes of benzene and isobutanol. In shaking the extract with 0.05N NaOH, PNP is quantitatively partitioned into the aqueous phase and can be determined spectrophotometrically at 410 nm. Aliquots of the solvent phase which now contains EPN and EPNO are evaporated to dryness at 120°C. One portion of the residue is treated with ethanol and alkaline hydroxylamine. The PNP liberated is equivalent to the total EPN -f EPNO. Another portion of the residue is dissolved in cyclohexane and shaken with alkaline hydroxylamine. EPNO, which has a greater reactivity and is more soluble in water, is rapidly decomposed to PNP and can be measured as above. EPN remains unaffected in the cyclohexane phase under these conditions. The value for EPN is calculated either by difference or, when necessary, by evaporating the cyclohexane and decomposing the residue with ethanol and hydroxylamine. The method is sensitive to 0.1 μmole EPN, EPNO, or PNP and can be used in the study of enzyme systems converting EPN and EPNO to PNP or EPN to EPNO. It can also be used in monitoring the preparation of EPNO from EPN by bromine water.