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-b ased dyes (either zwitterionic RH160, RH421, di-4-ANEPPS, or positivel y charged RH795, RH414, RH461) to phosphorylation of Na,K-ATPase from ATP or inorganic phosphate, and ouabain binding to phospho- or dephosp hoforms, have been characterized and compared in broken membrane prepa rations of the enzyme. Zwitterionic dyes were more sensitive to molecu lar events in the Na,K-ATPase reaction cycle than positively charged d yes, 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 i n the quantum yield of fluorescence. Computer modeling of dyes with id entical chromophore structure, and experimental characterization of th eir optical properties in bulk solvents, revealed two general trends: (1) the absorption maximum of the zwitterionic dye was blue-shifted wi th 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 d yes in the membrane depended on the presence of Na,K-ATPase. We sugges t, that (1) electrostatic interactions between the enzyme and the hydr ophilic headgroup of the dye by changing the charge of hydrophilic moi ety and thus modifying the net charge of the dye molecule cause both t he spectral shifts and the changes in the quantum yield, and (2) inter actions between the styryl dyes and the Na,K-ATPase depend on the conf ormational state of the enzyme.