68 



clairfy the role of receptor mediated uptake of ct-amanitin 

 conjugates. For this reason conjugates of a-amanitin and 

 the plant lectin, Concanavalin A, were synthesized. 



ADH-Con A Conjugates 



Synthesis 



Similar conditions to those used for the synthesis of 

 ADH-BSA were used to prepare ADH-Con A conjugates. The con- 

 jugates were purified after carbodiimide coupling by chrom- 

 atography on Sephadex G-75 (Figure 9) . The ADH-Con A eluted 

 in the void volume of the G-75 column and contained substan- 

 tial absorbance at 384nm and at 280nm (Figure 10) . From the 

 molar extinction coefficients of Con A and the azo moiety 

 it was determined that all of the Con A applied to the column 

 eluted in a single peak and contained a molar ratio of a- 

 amanitin to Con A of 0.67. 

 Characterization 



The ADH-Con A conjugates were first characterized with 

 respect to their inhibition of calf thymus RNA polymerase II. 



The results presented in Figure 11 generate an inhibition 



-9 

 constant of 186 x 10 M for calf thymus polymerase. The 



high K observed implies that a substantial reduction in the 

 affinity of a-amanitin for calf thymus RNA polymerase II 

 has occurred as a result of conjugation to Con A. Further- 

 more, as evidenced by the lack of binding of the ADH-Con A 

 to Sephadex G-75, these conjugates possessed little or none 

 of the ligand binding specificity of the native lectin. 



