Highly sensitive assay of okadaic acid using protein phosphatase and paranitrophenyl phosphate.
A colorimetric phosphatase-inhibition bioassay was developed for the quantitative measurement of okadaic acid (OA) the main diarrhetic toxin responsible for diarrhetic shellfish poisoning. The assay used an artificial substrate, paranitrophenylphosphate, and a semi-purified protein phosphatase PP2Ac containing extract prepared from rabbit muscle. Calibration dose-inhibition curves were constructed using standard OA and they permitted easy determination of the enzyme concentration Et in their linear portion. In the range of linearity, the slope increased when Et decreased, thus giving a detecting limit of 0.04 pmol in the reaction mixture (1 ml). The lowest assayable concentration of OA was 4 ng/ml in aqueous solutions and 40 ng/ml (i.e., 100 ng of OA per g of mussel tissue) in crude methanol mussels extracts. The intra and interassay coefficients of variation in the measurement of OA for the toxin spiked aqueous samples averaged, respectively, 7.7% and 3.7%, and interexperiments coefficients of variation for the toxin spiked mussel extracts averaged 4.6%. The presence of OA was ascertained by a method in which one assay was performed at two or three different levels of enzyme concentration. The rapidity, accuracy, reproducibility, specificity, and simplicity of the procedure provides a simple way to assay okadaic acid in buffered or complex solutions.
Simon
J F
JF
Laboratory of Cellular and Molecular Physiology, University of Caen, France.
Vernoux
J P
JP
D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D049872:Bivalvia / Q000201:enzymology; D002138:Calibration; D003124:Colorimetry; D004305:Dose-Response Relationship, Drug; D004988:Ethers, Cyclic / Q000032:analysis* / Q000494:pharmacology; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D016014:Linear Models; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism*; D019319:Okadaic Acid; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011817:Rabbits; D015203:Reproducibility of Results; D014874:Water Pollutants, Chemical / Q000032:analysis
D004988:Ethers, Cyclic; D007202:Indicators and Reagents; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D014874:Water Pollutants, Chemical; D019319:Okadaic Acid; C008644:nitrophenylphosphate; D010749:Phosphoprotein Phosphatases