52 

 as determined from the absorption spectra. Lowry protein 

 analysis of the conjugate yielded slightly lower values for 

 the amount of protein present than those derived from the 

 280nm absorbance. Since no interference with the Lowry 

 assay was noted from ADH added to known quantities of BSA, 

 it is presumed that the ADH conjugation induces a slight 

 increase in the 280nm absorption of the BSA. A factor of 

 1.18 times the 384nm absorbance was subtracted from the 

 280nm value to empirically correct for these differences. 

 Inhibition of calf thymus RNA polymerase II by the individ- 

 ual fractions obtained for G-75 chromotography gave addition- 

 al confirmation of the association of a-amanitin with BSA 

 for the ADH-BSA conjugate. 



The extent of protein to protein cross-linking result- 

 ing as a side reaction from carbodiimide coupling was esti- 

 mated by SDS-PAGE of the ADH-BSA. The relatively high pro- 

 tein concentrations (lOmg/ml) and high EDC concentration 

 (lOOmg/ml) used for conjugate synthesis led to significant 

 cross-linking, shown in Figure 5. Estimation of the area 

 under each peak of the scan of gel 1 indicated approximately 

 50% of the protein was in the monomeric form. Molecular 

 weight calibration with known protein standards (Figure 6) 

 demonstrated the cross-linked material to be in multiples 

 of BSA molecular weights (66,000 daltons) which would imply 

 that no fragmentation of the protein occurs during conjuga- 

 tion. 



