USING AFFINITY CAPILLARY ELECTROPHORESIS TO DETERMINE BINDING STOICHIOMETRIES OF PROTEIN LIGAND INTERACTIONS

We have developed a new method utilizing affinity capillary electropho resis (ACE) for the determination of binding stoichiometries in bioche mical systems. Using the same concentration of a ligand in the sample and the electrophoresis buffer, the appearance of an inverted peak cor responding to the free ligand in the resulting electropherogram provid es a criterion of binding of a ligand to its receptor protein. For bot h low (fast off rates) and high (slow off rates) affinity systems, ana lysis of the integration of free ligand peak in electropherograms as a function of the total concentration of a ligand in samples at constan t concentration of receptor protein yields the binding stoichiometry o f the ligand to the protein. Applications of this technique to studies of (i) the inhibition of carbonic anhydrases (CA, EC 4.2.1.1, from hu man and bovine erythrocytes) by 4-alkylbenzenesulfonamide 1, (ii) the interaction of a monoclonal antibody to human serum albumin (anti-HSA) with its antigen HSA, and (in) the binding of streptavidin (from Stre ptomyces avidinii) to biotin derivatives (monobiotinylated oligodeoxyr ibonucleotide 2, fluorescein biotin, or Lucifer Yellow biotin) yield s toichiometries of 1:1, 1:2, and 1:4, respectively. For multivalent, ti ght-binding systems, this ACE method can readily separate stable inter mediate species. This method is generally applicable to both tight- an d weak-binding systems, requires only nanograms of proteins and ligand s, involves no radioactive materials, and does not require changes in electrophoretic mobilities of receptor proteins upon binding with liga nds. It thereby provides a rapid, sensitive, and convenient method for measuring binding stoichiometries of ligands to proteins.