Cyanobacteria produce a class of hepatotoxins with over 150 variants, called microcystins (MC), that are especially harmful to mammals. In drinking water treatment, these compounds are partially oxidized, and little is known about the toxicity of the oxidation products. Oxidative pre-treatment with permanganate is effective in degrading these compounds via an attack on alkene or amide groups. Some products formed by this oxidation also have the potential to be toxic, specifically if the non-proteogenic amino acid (Adda) unique to microcystins is largely intact. Kinetic results for microcystin-leucine/arginine (MC-LA), a common and highly toxic microcystin congener, show that the primary oxidation products are C46H69N7O15 [M+H]+ =960.4925 and C46H67N7O14 [M+H]+=942.4819, both of which have isomeric forms that may contain an unmodified Adda moiety. The Adda group directly participates in inhibition of protein phosphatases, and it is also detected in the Adda-ELISA kit that is widely used in the drinking water industry. An alternative method of quantifying microcystins is by observing inhibition of their targets. Protein phosphatase inhibition assays (PPIA) have been developed using protein phosphatase 1 and 2A. Anecdotal evidence suggests that the Adda-ELISA may in some cases cross-react with oxidation products that do not exhibit protein phosphatase inhibition. Consequently, there is controversy over the use of this assay in drinking water treatment for microcystin detection because oxidation products may yield artificially high results. We describe purification schemes to isolate isomers of the oxidation products and assess their structure by ESI mass spectrometry (LC-qTOF). We have modified PPIA to measure MC-LA and assess the relative toxicity of the purified MC-LA oxidation products. The ability to accurately assign toxicity to these byproducts and identify inferences with the Adda-ELISA will ensure that treatment for these toxins is complete and effective.

Protein phosphatase inhibition assays (PPIA) for the evaluation of microcystin oxidation products

2019 Spring National Meeting

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