94 



reaction conditions, and pH 5 phosphate buffer in particular, 

 seem to preserve the native subunit structure of Con A. 



The nature of the chemical bond between the hippuric 

 acid residue and Con A was examined by exposure of the con- 

 jugates, prepared as described above, to hydroxylamine. The 

 conditions of exposure were such that hydrolysis of ester 

 type bonds, that may form as a side reaction during carbo- 

 diimide coupling (Carraway and Koshland, 1972; Timkovich, 

 1977), would be complete. The conjugate prepared in 

 phosphate buffer was 88% stable to hydroxylamine and the 

 preparation from the NaCl reaction contained 65% stable 

 bonds. These would presumably represent covalent peptide 

 linkages . 



The ligand binding activity of the two conjugates as 

 determined from the ability to bind to Sephadex G-75 and be 

 eluted with specific saccharide ligand is shown in Figure 19. 

 Complete retention of saccharide binding activity by this 

 criterion was demonstrated for both conjugates. They ad- 

 sorbed to the gel and were eluted by the Con A specific 

 ligand, . 1M D-glucose, in volumes identical to native Con A. 



A more quantitative determination of the ligand binding 



activity of the HA-Con A conjugates was made by determining 



the apparent association constant (K ) of each conjugate 



a 



for the chromogenic ligand, PNPM. Details of the method 

 are described in Materials and Methods and the results are 

 presented in Figure 20. At 22°C and under the conditions 

 of the assay used, native Con A had an affinity constant of 



