A novel spectroscopic titration method for determining the dissociation constant and stoichiometry of protein-ligand complex.
We offer a new titration protocol for determining the dissociation constant and binding stoichiometry of protein-ligand complex, detectable by spectroscopic methods. This approach neither is limited to the range of protein or ligand concentrations employed during titration experiment nor relies on precise determinations of the titration "endpoint," i.e., the maximal signal changes upon saturation of protein by ligand (or vice versa). In this procedure, a fixed concentration of protein (or ligand) is titrated by increasing volumes of a stock ligand (or protein) solution, and the changes in the spectroscopic signal are recorded after each addition of the titrant. The signal for interaction between protein and ligand first increases, reaches a maximum value, and then starts decreasing due to dilution effect. The volume of the titrant required to achieve the maximum signal changes is utilized to calculate the dissociation constant and the binding stoichiometry of the protein-ligand complex according to the theoretical relationships developed herein. This procedure has been tested for the interaction of avidin with a chromophoric biotin analogue, 2-(4'-hydroxyazobenzene)benzoic acid by following the absorption signal of their interaction at 500 nm. The widespread applicability of this procedure to protein-ligand complexes detected by other spectroscopic techniques and its advantages over conventional methods are discussed.
Wang
Z X
ZX
Biochemistry Department, North Dakota State University, Fargo 58105.
Kumar
N R
NR
Srivastava
D K
DK
D001360:Avidin / Q000378:metabolism*; D001391:Azo Compounds; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D008433:Mathematics; D008962:Models, Theoretical; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013816:Thermodynamics
D001391:Azo Compounds; D005456:Fluorescent Dyes; D008024:Ligands; D011506:Proteins; D001360:Avidin; C001413:HABA