Optimization of Sample Pretreatment Methods for the Applications of Glutamate Oxidase Reactor in Flow Injection Analysis of the Neurotoxic P -Odap in Grass Pea (Guaya)

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Addis Ababa Universty


In the determination of p-ODAP by flow injection analysis using a glutamate oxidase reactor , the major problem is that proteins and other macromolecules cause a rapid decay of the activity of the reactor when an extract of grass pea is directly injected into the reactor. In this work, off-line and on-line protein separation is described. In a preliminary study, a standard protein sample, bovine serum albumin (BSA), was treated with acidic alumina at different pH. Excess BSA was washed and the absorbance of the supernatant was taken at 280 nm. It was found that 84% of the protein was adsorbed on alumina at pH 7 (off-line separation). The recovery of p -ODAP was also studied at different pH by treating it with the alumina . Absorbance at 220 nm, before and after its treatment with the alumina revealed 100± 5% recovery at pH 7 . BSA and p -ODAP were combined and treated with alumina to check whether their coexistence has an effect on their interaction with alumina. Since P-ODAP may interfere with the measurement carried at 280 nm , the amount of the adsorbed protein was quantitated at 750 nm ( Lowry -Folin method) and found to be 85% which is in a good agreement with the result obtained when the measurement of BSA was carried alone (84% ). The strong anion exchanger (SAX), trimethylaminopropyl Isolute SAX(500-0010A), was employed for on-line separation of proteins. Of the injected standard protein and those from extracts of grass pea, 98% were retained by the sample clean-up column at a pH of 6.5 . This indicates that effective removal of proteins was achieved . The sample clean-up column has no significant effect in retaining P-ODAP and 98 % of the injected standard P -ODAP was recovered .