Production of 15N-labeled α-amanitin in Galerina marginata. (1st September 2015)

• Record Type: Journal Article
• Authors: Luo, Hong; DuBois, Brandon; Sgambelluri, R. Michael; Angelos, Evan R.; Li, Xuan; Holmes, Daniel; Walton, Jonathan D.
• Is Part Of: Toxicon. Volume 103(2015)
• Page Start: 60
• Page End: 64
• Publication Date: 2015-09-01
• Subjects: Amanita -- Galerina -- α-Amanitin -- Isotope labeling -- LC-MS -- NMR; Toxins -- Periodicals; Venom -- Periodicals; 615.9
• DOI: 10.1016/j.toxicon.2015.06.018
• Languages: English
• ISSNs: 0041-0101

Abstract

Abstract: α-Amanitin is the major causal constituent of deadly Amanita mushrooms that account for the majority of fatal mushroom poisonings worldwide. It is also an important biochemical tool for the study of its target, RNA polymerase II. The commercial supply of this bicyclic peptide comes from Amanita phalloides, the death cap mushroom, which is collected from the wild. Isotopically labeled amanitin could be useful for clinical and forensic applications, but α-amanitin has not been chemically synthesized and A. phalloides cannot be cultured on artificial medium. Using Galerina marginata, an unrelated saprotrophic mushroom that grows and produces α-amanitin in culture, we describe a method for producing 15 N-labeled α-amanitin using growth media containing 15 N as sole nitrogen source. A key to success was preparing 15 N-enriched yeast extract via a novel method designated "glass bead-assisted maturation." In the presence of the labeled yeast extract and 15 N–NH4 Cl, α-amanitin was produced with >97% isotope enrichment. The labeled product was confirmed by HPLC, high-resolution mass spectrometry, and NMR. Highlights: Efficient enrichment required growing Galerina with 15 N-ammonium chloride plus 15 N-labeled yeast extract. Enrichment of >97% was obtained in all 10 N atoms of α-amanitin. The labeled material will be useful for forensic and clinical applications.