24 



395 2 



is at 395nm (E = 1400cm /mMole) . Inhibition of calf 



thymus RNA polymerase II was determined for the peak fractions 

 and the ADGG-BSA conjugate was lyophilized and stored at -20°C, 

 ADGG-Con A 



The optimal reaction conditions for production of ADGG- 

 Con A conjugates were determined from a series of experiments 



• • • 14 14 



utilizing C-hippuric acid ( C-HA) as a free carboxyl group 



containing analog for ADGG. The optimal conditions for 



3 

 conjugation with Con A were verified in studies with H-DM- 



14 3 



ADGG. Details of the C-HA and H-DM-ADGG reactions are 



presented in later sections. The procedure described below 



represents a typical synthesis used for the production of 



ADGG-Con A conjugate. 



The Con A used for reaction with ADGG or hippuric acid 



was purified by affinity chromatography on Sephadex G-100 



followed by exhaustive dialysis versus water and lyophili- 



zation as described by Agrawal and Goldstein (1965) . 



Purified, lyophilized Con A was stored at -20°C until used. 



Prior to conjugation, Con A was dissolved to an approximate 



concentration of lOmg/ml in 0.01M phosphate buffer, pH 5 and 



allowed to remain at room temperature for 1 hour. The turbid 



solution was then clarified by centrif ugation at 10,000rpm 



(Beckman J-21B centrifuge; JA-20 rotor) for 30 minutes. The 



supernatant was filtered immediately prior to use through a 



. 2\i Millipore filter and the concentration determined by 



absorbancy at 280nm. Reactants were added in the following 



order to achieve final concentrations as listed: . Olymoles/ml 



