21 



were cooled to 0.2°C and lOray of NaNC- were added. Follow- 

 ing 10 minutes of occasional shaking, 18.5mg of a-amanitin 

 in 2.0ml cold pyridine were added. After 10 more minutes 

 at 4°C the reaction mixture was dried in vacuo , resolubi- 

 lized in 4.0ml 80% methanol and chromatographed on Sephadex 

 LH-20 in 80% methanol. Identification of the resulting 

 product, a-amanitin-diazobenzoylglycylglycine (ADGG) was 

 achieved by TLC , uv and NMR spectroscopy. 



A tritium labeled derivative of ADGG used in these 



studies was prepared and purified by Dr. J. F. Preston. The 



3 3 



derivative, H-demethyl-ADGG ( H-DM-ADGG) was synthesized 



by oxidation of ADGG with sodium periodate and subsequent 



3 

 reduction with H-NaBH . Purification by column chromato- 

 graphy on Sephadex LH-20 with various buffers to isolate the 

 product and remove exchangeable tritium resulted in a pro- 



o 



duct with a specific activity of 7.4 x 10 dpm/ymole. The 



H-DM-ADGG was determined to be essentially free from con- 

 taminants detectable by TLC and fluorography according to 

 the methods of Randerath (1970). The absorption spectra 

 and inhibition obtained for calf thymus RNA polymerase II as 

 well as TLC mobility indicated that it is identical to ADGG. 



Synthesis of a-Amanitin-Protein Conjugates 



ADH-DS A 



Conjugation to bovine serum albumin (BSA) was performed 

 by modifications of the methods presented by Faulstich and 

 Trischmann (1973) . The first step of the conjugation was 



