91 



under which a defined number of acid residues may be intro- 

 duced onto Con A by EDC. 

 Characterization 



For examination of the extent to which hippuric acid 

 conjugates of Con A retain lectin associated properties, 



two different conjugates were prepared utilizing the same 



14 

 concentration of ' C-HA described above. One was prepared 



in the presence of . 1M phosphate buffer, pH 5 and the other 

 in 0.1M NaCl, pH 7.2. Both were reacted with lOymole/ml of 

 EDC for 1 hour. After reaction they were dialyzed and sub- 

 jected to SDS-PAGE. The results are shown in Figure 18. 

 The upper gel contains molecular weight standards ranging 

 from 53,000 to 265,000 daltons. The next gel (2) is a 

 sample of native Con A and shows the typical 2 4,000 dalton 



subunit with a small amount of the naturally occurring 



14 

 fragments of Con A. Gel 3 contained C-HA-Con A conjugate 



prepared in phosphate buffer, pH 5. This conjugate prepar- 

 ation contained 2.4 residues of hippuric acid per mole of 

 Con A. The electrophoresis shows a lack of material with 

 a molecular weight greater than the Con A subunit. This 



would indicate that no cross-linking of the protein subunits 



14 

 has occurred. The bottom gel contained C-HA-Con A conju- 

 gate from the NaCl reaction. The molar ratio of hippuric 

 acid moieties to Con A for this conjugate was 9.6. This 

 conjugate contained some degree of cross-linked material, 

 estimated to be less than 8% of the total by scanning the 

 gels at 600nm on a recording spectrophotometer. Both 



