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Determination of protein‐ligand dissociation constants of their pH dependence by combined isoelectric focusing‐electrophoresis (titration curves): Binding of phosphorylases a and b to glycogen
Author(s) -
Ek Kristina,
Righetti Pier Giorgio
Publication year - 1980
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150010303
Subject(s) - chemistry , titration , dissociation constant , affinity electrophoresis , titration curve , isoelectric focusing , dissociation (chemistry) , ligand (biochemistry) , electrophoresis , covalent bond , analytical chemistry (journal) , chromatography , molecule , inorganic chemistry , biochemistry , affinity chromatography , organic chemistry , enzyme , receptor
Affino‐titration curves, a variant of conventional affino‐electrophoresis techniques, allow the simultaneous determination of protein‐ligand dissociation constants (K d ) and of their pH‐dependence in the pH range 3–10. If the ligand is a macromolecule, it is simply entrapped in the gel matrix; if it is a small molecule, it is covalently bound to the gel fibers. When a titration curve of a protein is run in presence of increasing amounts of ligand in the gel, the progressively decreasing mobilities, when plotted against the ligand molarity in the gel, can be used to calculate K d 's at any pH value. This technique has been used to measure the dissociation constants of glycogen with phosphorylases a and b, and their pH‐dependence.