Conjugation of Microcystins with Thiols Is Reversible: Base-Catalyzed Deconjugation for Chemical Analysis

Microcystins are potent cyclic heptapeptide toxins found in many freshwater cyanobacteria. Most microcystins contain an α,β-unsaturated amide that can react with thiol-containing 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.