Protein Phosphatase Inhibition Assay for Detection of Diarrhetic Shellfish Poison in Oyster

Graduate School, Chinese Academy of Sciences, Peping, Beijing, China
Chinese Journal of Analytical Chemistry (Impact Factor: 0.75). 03/2006; 34(3):283-287. DOI: 10.1016/S1872-2040(06)60014-5


AbstractA method based on protein phosphatase enzyme activity inhibition for monitoring diarrhetic shellfish poison (DSP) was used to analyze the DSP toxicity in three oyster samples. Based on the standard dose-effect curve developed with a series of okadaic acid (OA) standard solutions, the DSP toxicity of the three oyster samples collected was screened, and the results showed that OA and dinophysis toxins (DTXs) were not detected in the samples without hydrolyzation. However, the OA toxicity was detected in two of the hydrolyzed samples; the concentration of the OA was 1.81 and 1.21 μg OA eq./kg oyster, respectively.


Available from: Aifeng Li, Jun 12, 2014
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    • "One of the most promising methods that appeared recently for analysis of DSP toxins was the protein phosphatase inhibition assay based on the inhibition of the enzyme activity by OA and DTXs. PP1 and PP2A were the major forms of protein phosphatase, which catalyzed the phosphorylation on serine and threonine residues (Aifeng et al., 2006). "
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    ABSTRACT: A bienzyme electrochemical probe has been assembled and used to monitor the inhibition of the enzyme protein phosphatase-2A (PP2A) by okadaic acid (OA), taking advantage of the particular characteristics of a biochemical pathway in which PP2A is involved. This enzyme has significant activity toward glycogen phosphorylase a (PHOS a), which in turn catalyzes the conversion of glycogen to glucose-1-phosphate (G-1-P). In addition, PP2A is strongly inhibited by OA and its derivatives. Due to this combination of properties, PP2A was employed to develop an assay system involving a preliminary phase of off-line enzymatic incubations (OA/PP2A, PP2A/PHOS a, PHOS a/glycogen+phosphate). This off-line step was followed by the electrochemical detection of H2O2, which is the final product of two sequential enzymatic reactions: G-1-P with alkaline phosphatase (AP) producing glucose, then glucose with glucose oxidase (GOD) producing hydrogen peroxide. These two enzymes were coimmobilized on a nylon net membrane that was placed over an H2O2 platinum probe inserted into a flow injection analysis (FIA) system. During a first phase of the study, all analytical parameters were optimized. During a subsequent phase, the inhibition of PP2A enzyme by OA was evaluated. The calibration of the system shows a working range for detection of OA between 30 and 250 pg ml(-1). The total analysis time is the sum of 50 min for the off-line enzymatic incubations and 4 min for the biosensor response.
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