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Fluorescence Competition Assay
for the Assessment of ATP Binding to an Isolated Domain of Na+,
K+-ATPase
M. KUBALA1,2, J. PLÁŠEK1 ,
E. AMLER2
1Institute of Physics, Charles University Prague,
2Institute of Physiology, Czech Academy of Sciences,
Prague, Czech Republic
Received October 11, 2002
Accepted May 14, 2003
Summary
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
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