Α-Amanitin

α-Amanitin (alpha-Amanitin) is a cyclic peptide of eight amino acids. It is possibly the most deadly of all the amatoxins, toxins found in several species of the mushroom genus Amanita, one being the death cap (Amanita phalloides) as well as the destroying angel, a complex of similar species, principally A. virosa and A. bisporigera. It is also found in the mushrooms Galerina marginata, Lepiota subincarnata and Conocybe rugosa. The oral LD₅₀ of amanitin is 100 μg/kg for rats.

Unlike most cyclic peptides, amatoxins (and phallotoxins) are synthesized on ribosomes. The genes encoding the proprotein for α-amanitin belong to the same family as those that encode for phallacidin (a phallotoxin).

α-Amanitin is a selective inhibitor of RNA polymerase II and III but not I. This mechanism makes it a deadly toxin.

α-Amanitin can also be used to determine which types of RNA polymerase are present. This is done by testing the sensitivity of the polymerase in the presence of α-amanitin. RNA polymerase I is insensitive, RNA polymerase II is highly sensitive (inhibited at 1μg/ml), RNA polymerase III is moderately sensitive (inhibited at 10μg/ml), and RNA polymerase IV is slightly sensitive (inhibited at 50μg/ml).^{[citation needed]}

α-Amanitin is a highly modified bicyclic octapeptide consisting of an outer and an inner loop. The outer loop is formed by peptide bonds between a carboxyl terminus of an amino acid to the subsequent amino terminus of the next residue. The inner loop is closed by a tryptathionine linkage between 6-hydroxy-tryptophan and cysteine. In addition, α-amanitin is decorated with modified amino acid side chains (2S,3R,4R)-4,5-dihydroxy-isoleucine, trans-4-hydroxy-proline, which gives its high affinity for RNA polymerase II and III.

Early methods to detect alpha-amanitin included thin-layer chromatography (TLC). In most solvent systems used in TLC, alpha-amanitin and beta-amanitin would travel at different rates, thus allowing individual identification of each toxin. Another early method was the Meixner test (also known as the Wieland test), which would detect amatoxins, but also yielded false positives for some compounds, such as psilocin. Capillary zone electrophoresis was also developed, but was not adequately sensitive for clinical samples, but sufficient for mushroom extracts.

More recently, the use of high-performance liquid chromatography (HPLC) has become the preferred method, which allows for better resolution, reproducibility, and higher sensitivity. A range of detectors can be paired with HPLC, such as UV or mass spectrometry.

As early as the 1980s, antibody-based assays (immunoassays) were developed for amanitin (but more often recognize amatoxins as the antibodies cross-react with some of the congeners). The earliest immunoassays were radioimmunoassays and then enzyme linked immunosorbent assays (ELISAs). More, recently, in 2020, a monoclonal antibody-based lateral flow immunoassay (similar to a pregnancy test) has been developed that can quickly and selectively detect amatoxins in mushrooms and in urine samples.