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 p K 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°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 0.35°10 −3 s −1 resulting in a K d value of 0.4 μM 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 H 2 O was replaced by D 2 O. 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.