| Abstract
| - An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fastprotein liquid chromatography and partially characterized. The enzyme was purified 1871-fold with atotal yield of 40%. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) of thepure acid phosphatase resolved a single protein band that migrated to approximately 60 kDa.Nondenaturing SDS−PAGE electrophoresis revealed a single protein band around 120 kDa afterstaining with Coomassie Brilliant blue. Quantitative gel filtration chromatography estimated a nativemolecular mass of this enzyme to be 120 kDa. Thus, this acid phosphatase likely functions as ahomodimer, consisting of two similar 60 kDa subunits. An electrophoretic technique using theflourogenic substrate 4-methylumbelliferyl phosphate enabled visualization of an acid phosphataseactivity that corresponded to the protein band at 120 kDa on a nondenaturing PAGE gel. It wasdetermined that the acid phosphatase had a pH optimum of 6.0 at 25 °C. The enzyme activity appearedto be stable over a broad range of temperatures (10−40 °C) and in the presence of the metals Zn2+,Mn2+, and Mg2+ as well as the chelating agents ethylenedinitrilotetraacetic acid and ethylene glycoltetraacetic acid. It was shown that this acid phosphatase could hydrolyze a variety of physiologicalorganophosphate compounds including β-glycerophosphate, phosphoserine, adenosine triphosphate,adenosine diphosphate, adenosine monphosphate, and pyrophosphate. Furthermore, analysis usingcapillary electrophoresis demonstrated that this hydrolytic enzyme could transform a wide array oforganophosphate pesticides including S-2-ethylthioethyl O,O-dimethylphosphorothioate (demeton-S-methyl); S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethylphosphorodithioate (malathion); O,O-dimethylO-4-nitrophenyl (paraoxon); O,O,O,O-tetraethyldithiopyrophosphate (sulfatep); O-2-chloro-4-nitrophenyl O,O-dimethylphosphorothioate (dicapthon); and 2,2-dichlorovinyl dimethylphosphate (dichlorvos). Keywords: Acid phosphatase; organophosphates; phytometabolism; duckweed; Spirodela oligorrhiza
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