Fluoresceinyl-ethylenediamine-ouabain detects an acidic environment in the cardiac glycoside binding site of Na+/K+-ATPase

To probe the pH value in the microenvironment of the cardiac glycoside-binding site of Na+/K+-ATPase, pH-sensitive fluorescent derivatives of ouabain were synthesized. The fluoresceinyl derivative of ethylenediamino-ouabain (FEDO) had a pK(s) of 6.0 and showed a H+-dependent fluorescence change, when its ratio of excitation at 490 nm/450 nm was recorded at 530 nm. Binding of FEDO inactivated Na+/K+-ATPase at 37 degrees C and pH 7.25 in a slow time-dependent process under the conditions of backdoor phosphorylation with k(on) of 891 s(-1) M-1. The complex dissociated with k(off) of 0.35X10(-3) s(-1) resulting in a K-d value of 0.4 mu M for the FEDO . enzyme complex. Binding of FEDO was associated with a decrease of the excitatory fluorescence ratio at 490 nm/450 nm which could be used to convert this change into a pH value. A pH value of 5.1+/-0.2 was calculated to exist in the microenvironment of the FEDO . enzyme complex. This pH value was independent of the pH of the incubation medium used to form the FEDO . enzyme complex. Analysis of the accessibility of the fluorophore in the FEDO . enzyme complex to the dynamic quencher potassium iodide detected a decrease of the Stern-Volmer constant from 6.2 mM(-1) (free FEDO) to 1.5 mM(-1) (FEDO . enzyme complex) indicating thereby a limited accessibility of the fluorophore to anions. Analysis of the microenvironment of the fluorescein residue of the FEDO . enzyme complex by measurements of the anisotropy and the fluorescence half-life time revealed that both processes differed significantly when H2O was replaced by D2O. We conclude, therefore, that a pH of 5.1+/-0.2 exists in the vicinity of ouabain that is hidden in the depth of the receptor site when the ouabain . receptor complex has been formed.