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Microcystins are potent cyclic heptapeptide toxins
found in many freshwater cyanobacteria. Most microcystins
contain an α,β-unsaturated amide that can react with thiolcontaining
amino acids, peptides, and proteins in vivo and in vitro.
While soluble conjugates formed from small peptides can be
extracted and analyzed directly by LC−MS, microcystins
conjugated to proteins are analyzed after oxidative cleavage of
their Adda side chains, but information on which microcystin
analogues were present is lost. Observations during the development
of thiol-derivatization-based LC−MS methods for microcystin
analysis indicated that the reaction of thiols with
microcystins was reversible. The kinetics of deconjugation was investigated with mercaptoethanol as a model thiol to identify
suitable reaction conditions. A range of microcystins conjugated to mercaptoethanol, methanethiol, cysteine, and glutathione
were then successfully deconjugated, demonstrating the feasibility of releasing conjugated forms of microcystins for chemical
analysis. Reagents for removing the released thiols or for trapping the released microcystins increased the reaction rate.
Optimization of methodologies based on this reaction should increase the method’s utility for measuring free and conjugated
microcystins. The results also indicate that thiol-conjugated microcystins slowly release free microcystins, even at neutral pH,
with consequences for assessment of toxin exposure, metabolism, and trophic transfer. A range of other common natural and
environmental toxins, such as deoxynivalenol and acrylamide, also contain α,β-unsaturated carbonyl groups and can be expected
to behave in a similar manner. |
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