23 



Five milligrams of dried ADBH were dissolved in 4ml dry 

 trif luoroacetic acid, swirled for 1 minute and evaporated 

 to dryness at 40°C. Twenty milligrams of Con A (Sigma, 

 grade IV) dissolved in 2.5ml of water with 200mg EDC were 

 added to the ADH and reaction was carried out at room tem- 

 perature with intermittant mixing. A fine precipitate was 

 formed after 2 hours that settled out of the reaction mix- 

 ture. After 24 hours the reaction mixture was adjusted to 

 0.05% with respect to NH HCO and applied to a Sephadex G-75 

 column with reasonable care taken to leave the precipitate in 

 the reaction vessel. The conjugate ADH-Con A eluted with 0.05% 



NH.HCC- as a single peak followed by the unreacted ADH. Anal- 

 ly 



ysis of the individual fractions by absorption at 280nm (E, ° = 



lcm 



1.14) and 384nm and analysis of the pooled ADH-Con A peak by 

 inhibition of calf thymus RNA polymerase II was performed. 

 ADGG-BSA 



Conjugation of a-amanitin to free amino groups on BSA 

 was undertaken with the ADGG derivative by procedures similar 

 to those used to couple ADH. ADGG (3.34mg) was dissolved in 

 2.0ml of water along with 16.2mg of BSA. The pH was adjusted 

 to 7.2 with 0.1N NaOH and 200mg of EDC were added. After 

 2 4 hours of reaction at room temperature the product ADGG-BSA 

 was isolated by gel filtration on Sephadex G-75 with 0.05% 

 NH.HCO-. as eluant. The conjugate peak was analyzed by 

 absorption at 280nm and by the Lowry assay to determine 

 protein content. Alpha-amanitin content was determined from 

 the peak absorption of the diazonium moiety which for ADGG 



