Affinity titration curves Determination of dissociation constants of lectin-sugar complexes and of their pH-dependence by isoelectric focusing electrophoresis

Abstract

By performing electrophoresis perpendicular to a stationary pH gradient (pH-mobility curves) in polyacrylamide gels containing a specific ligand either covalently fixed or entrapped in the gel matrix, it is possible to measure dissociation constants ( K d) and their pH-dependence in the pH range 3.5–10. The present technique, called ‘affinity titration curves’, is an extension of ‘affinity electrophoresis’. This system has been applied to the study of the interaction between lectins and sugars: lectin from Ricinus communis seeds and α- d-galactose, and lectin from Lens culinaris seeds and α- d-mannose. The pH-dependence of K d values indicated a more rapid decrement of affinity of both lectins for their ligands at acidic pH as compared to alkaline pH. For both lectins, maximum affinity was found in the pH range 7–8. Since the ionic strength of focused carrier ampholytes is 100–200-times lower than in conventional electrophoresis, the K d values found by the present method are generally lower than the same values obtained by affinity electrophoresis.