Title: Purification and partial characterization of an acid phosphatase from Spirodela oligorrhiza and its affinity for selected organophosphate pesticides Hoehamer CF, Mazur CS, Wolfe NL Ref: Journal of Agricultural and Food Chemistry, 53:90, 2005 : PubMed
An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the pure 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 after staining with Coomassie Brilliant blue. Quantitative gel filtration chromatography estimated a native molecular mass of this enzyme to be 120 kDa. Thus, this acid phosphatase likely functions as a homodimer, consisting of two similar 60 kDa subunits. An electrophoretic technique using the flourogenic substrate 4-methylumbelliferyl phosphate enabled visualization of an acid phosphatase activity that corresponded to the protein band at 120 kDa on a nondenaturing PAGE gel. It was determined that the acid phosphatase had a pH optimum of 6.0 at 25 degrees C. The enzyme activity appeared to be stable over a broad range of temperatures (10-40 degrees C) and in the presence of the metals Zn2+, Mn2+, and Mg2+ as well as the chelating agents ethylenedinitrilotetraacetic acid and ethylene glycol tetraacetic acid. It was shown that this acid phosphatase could hydrolyze a variety of physiological organophosphate compounds including beta-glycerophosphate, phosphoserine, adenosine triphosphate, adenosine diphosphate, adenosine monphosphate, and pyrophosphate. Furthermore, analysis using capillary electrophoresis demonstrated that this hydrolytic enzyme could transform a wide array of organophosphate pesticides including S-2-ethylthioethyl O,O-dimethylphosphorothioate (demeton-S-methyl); S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethylphosphorodithioate (malathion); O,O-dimethyl O-4-nitrophenyl (paraoxon); O,O,O,O-tetraethyldithiopyrophosphate (sulfatep); O-2-chloro-4-nitrophenyl O,O-dimethylphosphorothioate (dicapthon); and 2,2-dichlorovinyl dimethylphosphate (dichlorvos).
