31 



column of Sephadex G-75 (2.4 x 40cm). After elution of one 

 column volume of PBS , 0.1M D-glucose was added to the eluant. 

 Monitoring of fractions for 280nm absorbancy was used to 

 determine the degree of binding to the gel matrix in refer- 

 ence to native Con A. 



Ligand affinity constant determination . The apparent 

 affinity constant for the ligand binding of selected conju- 

 gates was examined spectrophotometrically by the method of 

 Bessler et al. (1973). A chromogenic ligand, p-nitrophenyl 

 a-D-mannopyranoside (PNPM) generates a uv difference spectrum 

 following interaction with Con A the magnitude of which is 

 proportional to the affinity constant. A series of dilutions 

 of PNPM in 0.15M PBS were measured for absorbance at 317nm 

 as was the solution of Con A or Con A conjugate being examined. 

 A constant amount of the protein was then mixed with the 

 PNPM dilutions and 317nm absorbance redetermined. From these 

 data, the following parameters can be defined: P , concen- 

 tration of protein in protein and PNPM mixtures: D , concen- 

 tration of ligand (PNPM); AA_,_ =[A.,, 7 (protein) + A-.,., (ligand)] 



A , 7 (protein + ligand). A plot of -rj- versus . ,- for values 



I 

 of r , much greater than [P ] yields a y-intercept the re- 

 ciprocal of which equals AAmax or the change in absorbancy 

 when all ligand binding sites are saturated. From this value 

 the affinity constant can be determined by first calculating 

 the concentration of protein-ligand complex, [PD] from the 



relationship [PD] = -r— [P^]. The concentration of free 



^ AAmax t 



AA 

 ligand, [D] , is then calculated from [D] = [D ] - T^ max [ p t l • 



