Physiol. Res. 53: 109-113, 2004

Fluorescence Competition Assay for the Assessment of ATP Binding to an Isolated Domain of Na+, K+-ATPase


1Institute of Physics, Charles University Prague, 2Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic

Received October 11, 2002
Accepted May 14, 2003

An equation allowing estimation of the dissociation constant for binding of a non-fluorescent ligand to the enzyme is presented that is based on the competitive replacement of the ligand by its fluorescent analog. We derived an explicit formula for the probe fluorescence intensity, which is suitable for nonlinear least-squares analysis. We used this formula to evaluate the binding of ATP to the large cytoplasmic loop of Na+,K+-ATPase. The estimated value of KD (6.2 ± 0.7 mM) is comparable with the results from other laboratories for similar constructs obtained by a different method.

Key words
Competition • Dissociation constant • Fluorescence • Enzyme-ligand interaction • TNP-ATP

© 2004 by the Institute of Physiology, Czech Academy of Sciences