Using affinity capillary electrophoresis to determine binding stoichiometries of protein-ligand interactions.
We have developed a new method utilizing affinity capillary electrophoresis (ACE) for the determination of binding stoichiometries in biochemical systems. Using the same concentration of a ligand in the sample and the electrophoresis buffer, the appearance of an inverted peak corresponding to the free ligand in the resulting electropherogram provides a criterion of binding of a ligand to its receptor protein. For both low (fast off rates) and high (slow off rates) affinity systems, analysis of the integration of free ligand peak in electropherograms as a function of the total concentration of a ligand in samples at constant concentration of receptor protein yields the binding stoichiometry of 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 human 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 (iii) the binding of streptavidin (from Streptomyces avidinii) to biotin derivatives (monobiotinylated oligodeoxyribonucleotide 2, fluorescein biotin, or Lucifer Yellow biotin) yield stoichiometries of 1:1, 1:2, and 1:4, respectively. For multivalent, tight-binding systems, this ACE method can readily separate stable intermediate species. This method is generally applicable to both tight- and weak-binding systems, requires only nanograms of proteins and ligands, involves no radioactive materials, and does not require changes in electrophoretic mobilities of receptor proteins upon binding with ligands. It thereby provides a rapid, sensitive, and convenient method for measuring binding stoichiometries of ligands to proteins.
Chu
Y H
YH
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Lees
W J
WJ
Stassinopoulos
A
A
Walsh
C T
CT
D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods*; D005456:Fluorescent Dyes; D008024:Ligands; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism
D005456:Fluorescent Dyes; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D012709:Serum Albumin; D002256:Carbonic Anhydrases