Toward an understanding of the fluorescence intensity changes observed on fluorescein 5′-Isothiocyanate-Na+,K+-ATPase

Abstract

The fluorescence emission intensity between the Na(+), and the K(+) complex of Na(+),K(+)-ATPase, labeled with fluorescein 5'-isothiocyanate (FITC), differs by 30 to 40%. Experimental studies are carried out to elucidate the physical reasons which account this intensity difference. The dissociation constant of protolysis of the covalently bound FITC and its fluorescence decay times are determined in media of different ionic compositions and are compared with the corresponding properties of a synthetic model compound. The fluorophore bound to the protein is characterized by two decay times in the nanosecond range; the model compound, by a single one. The static fluorescence intensity changes are discussed on the basis of these results.

Inhibition and partial reactions of Na,K‐ATPase studied by fourier transform infrared difference spectroscopy

“…These intensity changes are related to a static quenching process. The fluorescence decay times as well as the pK a value of the covalently bound fluorophore remain unchanged upon inhibitor binding 11, 12. Therefore, the FITC fluorescence emission is measured in the presence of Mg 2+ with 0, 20, and 120 m M NaCl in dependence of time (Figure 4).…”