134 



Examination of the macromolecular composition of the 

 ADH-BSA conjugates by SDS-Page showed that the relatively 

 high concentrations of carbodiimide used for coupling caused 

 cross-linking of BSA into higher molecular weight complexes. 

 Covalent polymerization as a side reaction of carbodiimide 

 coupling was also reported by Timkovich (1977) to occur at 

 high carbodiimide and protein concentrations. The absence 

 of a specific ligand which interacts with BSA prevented any 

 quantitative measurement of the effects this cross-linking 

 may have on the recognition of BSA by cells. However, the 

 cross-linking may contribute to the reduction in binding 

 affinity for RNA polymerase seen with ADH-BSA. 



The effects of the ADH-BSA conjugates on cultured 

 mammalian cells were examined for three different cell lines. 

 These studies provided a comparison to the previously de- 

 scribed investigations of (3-amanitin-protein conjugates in 

 addition to establishing the methodology and preliminary 

 evidence necessary for examination of other a-amanitin pro- 

 tein conjugates. The toxicity of ADH-BSA for EL4 and M-7 

 CHO cells was approximately equal to that of free a-amanitin 

 on a molar basis of amanitin. This is surprising in view 

 of the greatly reduced affinity of ADH-BSA for RNA poly- 

 merase II. The enhanced toxicity observed of the conjugate 

 for AV3 cells indicates that either preferential uptake of 

 the conjugate, modification to a more toxic derivative, or 

 some combination of the two is occurring in these cells. 

 Since preferential uptake could result from specific 



