Evaluation of ligand induced relative change in the bimolecular quenching constant of protein fluorescence: a steady-state model.
Steady-state fluorescence quenching data for proteins in the presence of ligands can be obtained in such a fashion as to yield information about ligand induced changes in the bimolecular quenching constant, K+, without the necessity to determine the excited state lifetimes in separate measurements. We introduce Kr, the relative quenching constant assigned for the liganded protein (defined as the product of the bimolecular quenching constant characteristic of the enzyme-ligand complex and the average fluorescence lifetime in the absence of ligand and quencher) which reports, in comparison with the Stern-Volmer constant (KSV) assigned to the free protein, about the relative change in the dynamic exposure of the proteins fluorophore. Regardless of whether the protein is saturated with the ligand or not, the steady-state fluorescence data obtained with ligand and quencher concentrations as independent variables can be plotted to obtain linear relationships from which the values for Kr and KSV can be calculated.
Somogyi
B
B
Department of Biophysics, University Medical School of Debrecen, Hungary.
Matkó
J
J
Rosenberg
A
A
D004798:Enzymes / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D008024:Ligands; D008962:Models, Theoretical; D011506:Proteins; D013816:Thermodynamics
D004798:Enzymes; D008024:Ligands; D011506:Proteins