BackgroundCarnivorous marine snails, sp. Conus, produce venom peptides, known as conotoxins, which target voltage-gated and ligand-gated ion channels. One family, known as the a-conotoxins, specifically antagonizes different isoforms of nicotinic acetylcholine receptors (nAChRs). a-conotoxins are now widely used in neuroscience to study nAChRs that modulate neurotransmitter release. Although originally purified by venom, conotoxins may now be prepared by total chemical synthesis.ProblemThe a-conotoxins contain two disulfide bonds that must be oxidized for biological activity. Previously, we have synthesized a-conotoxins using solid-phase Fmoc chemistry with orthogonally protected cysteines to direct the subsequent folding. However, a common problem encountered during disulfide formation is undesired oxidation of amino acid side chains. Such is the case with conotoxin Ex1.7.MethodsEx 1.7 is a novel conotoxin containing amino acid side chains identified by gene cloning. The methionine at position 10 was oxidized when using our standard methods. Instead, glutathione was used as a mechanism to accelerate the disulfide bond formation and prevent prolonged exposure to atmospheric oxygen typically associated with the unwanted oxidation of methionine. The peptide was initially dissolved in 450 �l of buffer. The reaction was then initiated by adding 50 �l of glutathione. The reaction was allowed to proceed for 5, 10, 20, 40, or 80 minutes with progress monitored by HPLC.ResultsUpon completion of the reaction, three main products were obtained. These products will be subjected to mass spectrometry to confirm whether the three expected disulfide isomers were obtained without the oxidation of the methionine.ConclusionEquilibrium oxidation by glutathione may be an effective method for obtaining a-conotoxins with functional disulfide bonds and absent, unwanted oxidation of other sensitive side chains.

Avoiding unwanted oxidation of methionine using glutathione for disulfide bond formation

248th National Meeting (2014)

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