Detectionof Cyanobacterial Peptide Toxins by a Non-Radioactive Protein PhosphataseInhibition Assay

ReportNo WSAA 105

February 1996




Cyclicpeptide hepatotoxins produced by certain cyanobacteria (blue-green algae) are amajor concern to water authorities worldwide. These toxins can cause acute orchronic health effects in humans and animals if consumed. Consequently theirearly detection in waters used for drinking is highly desirable so thatmeasures to minimise or prevent exposures can be implemented, eg, introductionof an alternative water supply.


Thisreport assesses the performance of a colorimetric enzyme-based assay to detectcyclic peptide hepatotoxins by comparing it with an established technique ofhigh performance liquid chromatography (HPLC). The enzyme assay is based on theproperty of these hepatotoxins, namely microcystins produced by species of Microcystis and nodularin produced by Nodularia spumigena, to inhibit enzymesresponsible for the dephosphorylation of intracellular phosphoproteins. Anassay already exists for the determination of peptide hepatotoxins but itinvolves the use of a 32P radiolabelled phosphorylated proteinsubstrate. The radiolabelled protein phosphatase assay has several advantagesover HPLC, namely:


        lower detection limit


        measures all inhibitors of protein phosphatase including toxins whichmay not be detected by HPLC


        few toxin standards are available for use in quantification by HPLC.The phosphatase assay overcomes this limitation by measuring total toxicityrather than individual toxin concentrations


Howeverthe radiolabelled assay has a major disadvantage in that it requires the use ofa hazardous radioisotope which is relatively short-lived. Consequently a methodfor the determination of protein phosphatase inhibition based on thecolorimetric measurement of released orthophosphate was developed. Besides theadvantage of not requiring a radioactive substrate, it requires comparativelyless equipment and is also potentially cheaper. In addition, because of itspossible simplicity, it has the potential to be developed as a field kit.


Thecolorimetric based enzyme assay was tested for precision and the ability todetect toxins. Toxins were spiked into several natural waters with variouscharacteristics, eg. high colour and high turbidity, and the recoveriesdetermined. As large amounts of phosphorus in the sample will interfere in theanalysis, the method was developed to overcome this problem. Several toxic andnon-toxic samples of bloom material collected from the field were also examinedby both methods as well as by mouse bioassay.


Thecolorimetric assay showed promise with some consistency between the resultsobtained with that assay and those obtained by HPLC. It had a low detectionlimit, 0.2nM (approximately 0.2 mg/L) for the HPLCprocedure. However considerably more work needs to be done in comparing it withaccepted conventional procedures (both a radiolabelled procedure and HPLCanalysis), and to make it more practical.


Copiesof the Report are available from WSAA, price $A40. Orders may be placed throughthe Bookshop at or by email to