18 



polysaccharides and the mixture was filtered through 

 scintered glass. The resulting filtrate was flash evapora- 

 ted to near dryness, resolubilized in water and extracted 

 three times with 3 volumes of anhydrous ether. The final 

 extract was made 50% with respect to methanol (spectral 

 grade) and subjected to chromatography on Sephadex LH-20 

 in 50% methanol. The a-amanitin containing fractions were 

 identified by TLC on silica gel G with methyl ethyl ketone: 

 methanol (1:1) and detection after spraying with 2% methanolic 

 t-cinnamaldehyde and exposing to HC1 vapors (Wieland et al., 

 1949; Sullivan et al., 1965). The characteristic R f and 

 violet color served to identify amanitin containing fractions. 

 The peak fractions were combined, flash evaporated to dry- 

 ness, resolubilized in water and chromatographed on Biogel 

 P-2 in water. After concentration of pooled a-amanitin 

 fractions from this step, final purification was obtained by 

 chromatography on Sephadex LH-20 with water. The resulting 

 a-amanitin was characterized as to purity on the basis of 

 TLC mobility with two different solvent systems (methanol: 

 methyl ethyl ketone (1:1), n-butanol racetic acidtwater 

 (4:1:1), ultraviolet (uv) and visible absorption spectra, 

 and nuclear magnetic resonance (NMR) spectroscopy. All 

 samples prepared proved identical by these criteria to a 

 crystalline a-amanitin standard obtained from Th. Wieland, 

 Max Planck Institute for Medical Research, Heidelberg, 

 Germany. These purification procedures were developed from 

 procedures previously published (Faulstich, et al., 1973; 



