SECTION II 

 MATERIALS AND METHODS 



Modification of a-Amanitin for Conjugation to Proteins 



Isolation and Purification 



All chemicals used throughout these investigations 

 were of reagent quality and were used without further 

 purification unless noted otherwise. The primary commercial 

 suppliers were Scientific Products (Mallinkrodt) and Sigma 

 Chemical Co. Water for all procedures was deionized and 

 glass distilled. 



The a-amanitin used for these studies was obtained 

 primarily from specimens of Amanita suballiacea collected 

 from the Gainesville, Florida , area. Individual specimens 

 were collected, identified on the basis of gross morpho- 

 logical characteristics according to published descriptions 

 (Miller, 1977; Murrill, 1941; Murrill, 1948) and pooled for 

 extraction of a-amanitin. After thorough washing with 

 running tap water, the specimens were coarsely chopped and 

 combined with methanol to an approximate final concentration 

 of 50% methanol (v/v) . This crude slurry was shaken on a 

 rotary shaker at lOOrpm and 22°C for 24 hours, filtered 

 through Whatman #1 filter paper and the crude methanolic 

 extract was flash evaporated to a thick syrup. Ten volumes 

 of ice cold 100% methanol were added to precipitate 



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