Characterizing protein conformational transitions of Na, K-ATPase with antibodies by fluorescence spectroscopy

Stationary and time-resolved fluorescence of FITC-Na,K-ATPase is investigat ed as a function of pH in the presence of different ligands, cations, and t he monoclonal anti-FITC antibody 4-4-20. The binding of K+ and of the antib ody leads to the same decreased fluorescence intensity level. Antibody bind ing is observed only under conditions where the enzyme exists in the confor mational state F-1, and not in the form of the Na+ or K+ complex or when it is phosphorylated with inorganic phosphate in the presence of Mg2+. For th e interpretation of the results it is shown that the fluorophore is not ess entially affected by an acidity change of the bound dye, so that pK variati ons responsible for the observed intensity changes can be excluded in favor of a static quenching process