Detection of Cyanobacterial Peptide Toxins by a Non-Radioactive Protein Phosphatase Inhibition Assay

This report was produced for the Urban Water Research Association of Australia, a now discontinued research program.

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Detection of Cyanobacterial Peptide Toxins

Report No UWRAA 105

February 1996


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

This report assesses the performance of a colorimetric enzyme-based assay to detect cyclic peptide hepatotoxins by comparing it with an established technique of high performance liquid chromatography (HPLC). The enzyme assay is based on the property of these hepatotoxins, namely microcystins produced by species of Microcystis and nodularin produced by Nodularia spumigena, to inhibit enzymes responsible for the dephosphorylation of intracellular phosphoproteins. An assay already exists for the determination of peptide hepatotoxins but it involves the use of a 32P radiolabelled phosphorylated protein substrate. The radiolabelled protein phosphatase assay has several advantages over HPLC, namely:

  • lower detection limit
  • measures all inhibitors of protein phosphatase including toxins which may 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 toxicity rather than individual toxin concentrations

However the radiolabelled assay has a major disadvantage in that it requires the use of a hazardous radioisotope which is relatively short-lived. Consequently a method for the determination of protein phosphatase inhibition based on the colorimetric measurement of released orthophosphate was developed. Besides the advantage of not requiring a radioactive substrate, it requires comparatively less equipment and is also potentially cheaper. In addition, because of its possible simplicity, it has the potential to be developed as a field kit.

The colorimetric based enzyme assay was tested for precision and the ability to detect toxins. Toxins were spiked into several natural waters with various characteristics, eg. high colour and high turbidity, and the recoveries determined. As large amounts of phosphorus in the sample will interfere in the analysis, the method was developed to overcome this problem. Several toxic and non-toxic samples of bloom material collected from the field were also examined by both methods as well as by mouse bioassay.

The colorimetric assay showed promise with some consistency between the results obtained with that assay and those obtained by HPLC. It had a low detection limit, 0.2nM (approximately 0.2 mg/L) for the HPLC procedure. However considerably more work needs to be done in comparing it with accepted conventional procedures (both a radiolabelled procedure and HPLC analysis), and to make it more practical.

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