25 



Con A, 1. Oymole/ml ADGG and sufficient 0.1M phosphate buffer, 

 pH 5 to give a final concentration of of 0.01M phosphate. The 

 reaction was initiated by addition of 50vil of lmmole/ml EDC 

 in water per milliliter of reaction mix final volume. The 

 reaction was allowed to proceed at room temperature for 12 

 hours after which time it was adjusted to 0.15M with respect 

 to NaCl and applied to a small column of Sephadex G-100 in 

 0.15M phosphate buffered saline, pH 7.4 containing . ImM 

 maganese and calcium (PBS ) . The column bed volume was 

 generally 2 to 2.5 times the reaction mix volume. The 

 reaction mixture was allowed to enter the column at a rate 

 of approximately . lml/minute . Unreacted ADGG emerged from 

 the column first as essentially 100% of the Con A as ADGG- 

 Con A conjugate bound to the gel. After elution with 10 col- 

 umn volumes of PBS , 0.1M D-glucose was added to the eluant 

 causing the displacement of ADGG-Con A in a single well-de- 

 fined band. The conjugate was characterized by absorption 

 at 280 and 395nm and dialyzed extensively against 0.15M PBS , 

 pH 7.4 at 3-5°C. ADGG-Con A conjugates were stored at 3°C 

 and were used within three weeks of their synthesis. During 

 this period of time essentially no change in protein concen- 

 tration or absorbance spectra were noted. 

 3 H-DM-ADGG-Con A 



Verification of the optimal conditions for conjugation 

 of ADGG and Con A and production of a tritium labeled Con A 

 conjugate for cell binding studies were performed with 

 H-DM-ADGG. Details of the individual experiments are 



