133 



lack of a free carboxyl group on a-amanitin required that 

 it first be chemically modified to permit conjugation to 

 proteins via reaction with water soluble carbodiimides . 

 Diazotization of a-amanitin to an aromatic moiety contain- 

 ing a six carbon spacer molecule and a terminal free 

 amino group (Faulstich and Trischmann, 1973) provided the 

 ADH derivative that was readily purified by gel chromatog- 

 raphy and identifiable by its characteristic spectral 

 absorption and TLC mobility. Conjugation to BSA via 

 carbodiimide reaction provided covalently linked conjugates 

 of a-amanitin and BSA as evidenced by co-elution of protein 

 and 384nm absorbance of the azo moiety from Sephadex G-75. 

 Quantitation of the inhibition of calf thymus RNA polymer- 

 ase II by the ADH-BSA conjugates in comparison to free ADH 

 or a-amanitin (Figure 7) demonstrated that modification of 

 a-amanitin to the ADH derivative had little effect on its 

 binding to polymerase. Both ADH and a-amanitin have simi- 

 lar K values and are noncompetitive inhibitors. Conjuga- 

 tion to BSA, however, resulted in a 38-fold decrease in 

 binding affinity, most likely as a result of steric 

 hinderance rather than a conformational alteration of a- 

 amanitin. The lack of convergence of the three lines gen- 

 erated with different VIP concentrations implies that con- 

 jugation to BSA not only decreases binding affinity but 

 alters the nature of the inhibition from the strictly non- 

 competitive type. 



