Limitations in linearized analyses of binding equilibria: binding of TNP-ATP to the H4-H5 loop of Na/K-ATPase.
Binding of TNP-ATP [2',3'- O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, a fluorescent analogue of ATP] to the K605 protein was studied. This is an isolated N-domain in the cytoplasmic loop of the Na/K-ATPase alpha-subunit, lying between membrane-spanning segments 4 and 5 (sequence L(354)-I(604)). A titration equation is derived that explicitly makes it possible to relate the fluorescence of TNP-ATP and K605 solutions to total probe concentration in the sample. Using this, it is possible to obtain the value of the dissociation constant from the titration experiment without resorting to the Scatchard plot, which is far from optimal from the statistical point of view. Using the new formula with non-linear regression analysis, a value of the dissociation constant K(D) for TNP-ATP binding to the K605 protein of 3.03 +/- 0.28 microM at 22 degrees C was obtained. A series of fits to simulated data with added noise demonstrated clearly the advantage of non-linear regression using the new formula over the commonly employed linear regression using the Scatchard plot. The procedure presented is generally applicable to binding assays using changes in the fluorescence of ligands on binding.
Kubala
M
M
Institute of Physiology, Czech Academy of Science, Vídenská 1083, 142 00 Prague, Czech Republic.
Plásek
J
J
Amler
E
E
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry*; D000465:Algorithms; D001665:Binding Sites; D003198:Computer Simulation; D005456:Fluorescent Dyes; D007700:Kinetics; D016014:Linear Models; D008956:Models, Chemical; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase