88 



and EDC concentrations were examined. Carbodiimide 

 coupling was performed in the presence of . 1M phosphate 

 buffers and in all cases higher concentrations of EDC 

 (lOOymole/ml versus lOymole/ml) gave greater incorporation. 

 Both the rate of reaction and maximal amount of labeling 

 were increased. For a given concentration of EDC, the 

 lowest pH examined, pH 5, proved optimal with an EDC con- 

 centration of 10ymole/ml resulting in the incorporation of 



3 

 10 cpm per O.OOlumole Con A. An EDC concentration of 



3 

 lOOpmole/ml yielded a maximum of 3.3 x 10 cpm per O.OOlymole 



Con A at the same pH. Increasing the pH to 6 or 7 caused 

 a decrease in the rate and maximum level of incorporation. 

 Under all conditions examined here, the reactions were 

 essentially complete by 2 hours. 



The effects of the presence of NaCl, an important 

 parameter affecting the confirmations of Con A, at two 

 different EDC concentrations are presented in Figure 17. 

 No effect on the rate or extent of reaction was observed 

 in the presence of . 1M NaCl in comparison to the aqueous 

 reaction. Increasing the EDC concentration 10-fold resulted 

 in an approximate 5-fold increase in the maximum amount 

 of incorporation as well as an increase in the reaction rate. 

 The reactions in the absence of phosphate buffer took much 

 longer to go to completion (18-24 hours versus 2 hours) and 

 attained 10-fold greater maximal values than for the cor- 

 responding reaction at pH 5 in phosphate buffer. These data 

 would argue that phosphate is providing a controlled situation 



