Fluorescent styryl dyes as probes for Na,K-ATPase reaction mechanism: significance of the charge of the hydrophilic moiety of RH dyes.

The fluorescence responses of a series of potential-sensitive styryl-based dyes (either zwitterionic RH160, RH421, di-4-ANEPPS, or positively charged RH795, RH414, RH461) to phosphorylation of Na,K-ATPase from ATP or inorganic phosphate, and ouabain binding to phospho- or dephosphoforms, have been characterized and compared in broken membrane preparations of the enzyme. Zwitterionic dyes were more sensitive to molecular events in the Na,K-ATPase reaction cycle than positively charged dyes, but the net charge did not affect the sensitivity of the dyes to a transmembrane electric field. The major part of the response of the zwitterionic dyes to formation of phosphoenzymes was due to a change in the quantum yield of fluorescence. Computer modeling of dyes with identical chromophore structure, and experimental characterization of their optical properties in bulk solvents, revealed two general trends: (1) the absorption maximum of the zwitterionic dye was blue-shifted with respect to the positively charged dye; (2) the quantum yield of the zwitterionic dye was higher and the fluorescence lifetime was longer than that for the positively charged dye. Spectral properties of the dyes in the membrane depended on the presence of Na,K-ATPase. We suggest, that (1) electrostatic interactions between the enzyme and the hydrophilic headgroup of the dye by changing the charge of hydrophilic moiety and thus modifying the net charge of the dye molecule cause both the spectral shifts and the changes in the quantum yield, and (2) interactions between the styryl dyes and the Na,K-ATPase depend on the conformational state of the enzyme.