138 

 by the protein amino group, should be increased by running 



the reaction at lower pH where amino groups would be the 



14 

 rate limiting factor. Reaction of C-HA and Con A in 



dilute phosphate buffer solution at pH 5 , 6 or 7 resulted 

 in increased conjugation with lower pH . For a given pH , 

 increasing the concentration of carbodiimide 10-fold in- 

 creased the rate and extent of reaction. When the identi- 

 cal reaction was run at pH 7 in 0.1M NaCl, the extent of 

 conjugation was increased over the equivalent reaction in 

 phosphate buffer at pH 5 approximately 10-fold. Addition- 

 ally, the reactions in phosphate buffer reached completion 

 within 2 hours whereas without phosphate, the reaction took 

 18-24 hours to reach completion. These results indicate 

 that phosphate is limiting the rate and extent of reaction 

 and appears to provide a controlled condition for the con- 

 jugation of a limited number of acid residues to Con A. 



14 



C-HA-Con A conjugates prepared in either NaCl, pH 7 



or phosphate buffer, pH 5 were analyzed for protein cross- 

 linking by SDS-PAGE. In contrast to the ADH-BSA conjugates, 



a barely detectable amount of cross-linked material was 



14 

 observed for both " C-HA-Con A conjugates which represented 



less than 8% of the total material. This is undoubtedly a 



direct result of the reduction of carbodiimide concentration 



by 10-fold over that used for the ADH-BSA conjugates. The 



pH 5, . 1M phosphate buffer condition with ratios of Con A 



to HA to EDC of 1:100:1000 seem to be optimal for production 



of conjugates with minimal alteration of gross molecular 



properties. 



