27 



14 



C-HA-Con A 



Evaluation of the optimal reaction conditions for 



carbodiimide mediated coupling of free carboxyl containing 



derivatives of a-amanitin to Con A was performed with the 



14 

 use of ' C-hippuric acid as an analog. The reaction of 



14 



C-HA and Con A was examined under a number of different 



conditions. Reactions were generally carried out in a final 



volume of 1.0ml at room temperature. Procedures for sampling 



the reaction at various time points were those previously 



3 

 described for H-DM-ADGG reactions. The reaction buffer 



was either 0.1M sodium phosphate, pH 5, 6 or 7, or pH 7.2 



NaCl, 0.01 or 0.1M containing either 0.1 or 0.01M CaCl„ and 



MnCl_, respectively. Con A, (0 . 001ymole/ml) and 1.0ymole/ml 



14 5 



of C-carboxyl hippuric acid (8.5 x 10 dprn/pmole, ICN 



Chemical and Radioisotope Division) were reacted with either 



10 or lOOymoles of EDC in the buffer systems described above. 



HA-Con A 



14 

 Conjugates of hippuric acid and Con A without C label 



were prepared for analysis of the retention of lectin charac- 

 teristics by the HA-Con A conjugates. Two different reaction 

 conditions were chosen for these studies. Reaction mixtures 

 containing 0. OOlymole/ml Con A, O.lymole/ml HA and lOumole/ml 

 EDC were prepared in either . 1M phosphate buffer, pH 5 or 

 0.1M NaCl, pH 7. The reactions were incubated at room tem- 

 perature for 2 hours followed by overnight dialysis against 

 0.15M PBS at 5°C to remove unreacted components. The 

 resulting HA-Con A conjugates designated HA-Con A (PO.) and 

 HA-Con A (NaCl) were used without further purification. 



