K. Shimura et K. Kasai, DETERMINATION OF THE A AFFINITY CONSTANTS OF PEA LECTIN FOR NEUTRAL SUGARS BY CAPILLARY AFFINOPHORESIS WITH A MONOLIGAND AFFINOPHORE, Journal of Biochemistry, 120(6), 1996, pp. 1146-1152
Affinophoresis is a type of affinity electrophoresis in which an affin
ophore, a conjugate of an affinity ligand and a multiply charged solub
le matrix, causes a change in migration velocity of proteins which hav
e a specific affinity for the ligand. A monoligand affinophore bearing
a mannoside was prepared by coupling iodoacetylated p-aminophenyl alp
ha-D-mannoside to the free thiol group of N-succinylated glutathione,
and used for the affinophoresis of pea lectin in a capillary, The elec
trophoretic mobility of pea lectin towards the anode increased in the
presence of the affinophore as a function of its concentration in a ma
nner that is described by the equation for affinity electrophoresis. A
nalysis of the suppression of the affinophoresis on the addition of ne
utral sugars to the system allowed the determination of the dissociati
on constants of the lectin for these neutral sugars, The dissociation
constants obtained on affinophoresis agreed well with the values in th
e literature, The preparation of a monoligand affinophore for ligands
bearing an amino group should facilitate the application of this type
of microscale analysis (0.14 ng of protein for each run) to protein li
gand interactions.