137 



decrease in the affinity for calf thymus RNA polymerase for 



-9 

 the a-amanitin portion of the conjugate (K = 127 x 10 M) . 



As was seen for the ADH-BSA conjugates, conjugation also 



altered the nature of the inhibition from the strictly non- 



-9 

 competitive. Since free ADGG possessed a K of 6.9 x 10 M, 



not dramatically different from free a-amanitin, it appears 



likely that the reduction in binding affinity of the ADGG- 



Con A conjugate is due to steric hindrance by the protein 



portion of the conjugate. In any case, the ADGG conjugated 



to Con A is still a potent inhibitor of RNA polymerase II 



in vitro . 



Hippuric Acid-Con A Conjugates 



14 

 The use of ' C-hippuric acid as a free carboxyl group 



containing analog to ADGG was intended to define those condi- 

 tions of carbodiimide mediated coupling to Con A that would 

 result in the introduction of a defined number of acid moie- 

 ties onto Con A with minimal perturbation of the basic lec- 

 tin structures and ligand binding activities. All of the 

 previous conjugations had been carried out at pH 7 in 

 water with moderately high concentrations of EDC. The 

 proposed mechanism of carbodiimide coupling (Hoare and 

 Koshland, 1967; Carraway and Koshland, 1972) had as a first 

 step the formation of a carbodiimide-carboxyl group adduct 

 in the form of an O-acylisourea. A stable product is 

 formed by the nucleophilic displacement of the carbodiimide 

 group with subsequent formation of a piptide bond. The 

 specificity of nucleophilic attack, which in this case is 



