29 



for 12 hours (Carraway and Koshland , 1972). The remaining 

 TCA precipitable activity in comparison to a nonhydroylzed 

 control sample was used as an indicator of covalent bonding. 

 Protein-protein cross-linking . The extent of protein 



to protein cross-linking induced during the carbodiimide 



14 

 conjugation was evaluated for ADH-BSA and C-HA-Con A 



conjugates by sodium dodecylsulf ate-polyacrylamide gel 

 electrophoresis (SDS-PAGE) according to the method of Weber 

 and Osborn (1969) . Electrophoresis was performed with 5% 

 acrylamide disc gels run at 8 milliamp per gel. Visuali- 

 zation of the bands was achieved by staining with Commassie 

 brilliant blue (0.1%) in 50% TCA for 1 hour at 37°C (Laemmli, 

 1970) . Diffusion destaining with multiple changes of 7% 

 acetic acid was carried out to remove dye not associated 

 with protein. 

 Inhibition of Calf Thymus RNA Polymerase II 



Calf thymus RNA polymerase II was purified through DEAE 

 cellulose chromatography according to the method of Kedinger, 

 et al. (1975) and stored in liquid N„. A single preparation 

 of enzyme used for all studies presented here proved to be 

 extremely stable in liquid N_ over a four year period. 

 Assay of the inhibition of calf thymus RNA polymerase II 

 was performed according to the procedures developed by Preston, 

 et al. (1975) . The reaction mixture used was that described 

 by Cochet-Meilhac and Chambon (1974) . The inhibition assays 

 were performed as follows: To the bottom of a new 10 x 75mm 

 test tube was added 0.01ml of each inhibitor concentration 



