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 (Kd) 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 alpha-D-galactose, and lectin from Lens culinaris seeds and alpha-D-mannose. The pH-dependence of Kd 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 Kd values found by the present method are generally lower than the same values obtained by affinity electrophoresis.
Affinity titration curves: determination of dissociation constants of lectin-sugar complexes and of their pH-dependence by isoelectric focusing electrophoresis / K. Ek, E. Gianazza, P.G. Righetti. - In: BIOCHIMICA ET BIOPHYSICA ACTA. PROTEIN STRUCTURE. - ISSN 0005-2795. - 626:2(1980 Dec 16), pp. 356-365.
Affinity titration curves: determination of dissociation constants of lectin-sugar complexes and of their pH-dependence by isoelectric focusing electrophoresis
E. GianazzaSecondo
;
1980
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 (Kd) 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 alpha-D-galactose, and lectin from Lens culinaris seeds and alpha-D-mannose. The pH-dependence of Kd 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 Kd values found by the present method are generally lower than the same values obtained by affinity electrophoresis.Pubblicazioni consigliate
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