Interaction of 8-Anilino-1 -naphthalenesulfonate with Rod Outer Segment Membrane

The interaction between bovine rod outer segment (ROS) membrane and a fluorescent probe, 8-anilino-lnaphthalenesulfonate (Ans), has been studied to investigate the visual excitation process and structural aspects of the membrane. Binding of the probe to the membrane is indicated by its fluorescence enhancement and a blue shift of the emission band. When ROS membrane is bleached, a further increase in fluorescence and blue shift can be observed. These phenomena are attributed to local conformational changes in the opsin part of the molecule upon bleaching. The possibility of energy transfer from bound probe to 11-di-retinal has been excluded by lifetime measurements. Ans binds to both lipids and rhodopsin. The relative contribution to fluorescence of Ans bound to protein and lipid was assessed on the basis of fluorescence lifetime and steady-state emission spectra. The lifetime spectrum of lipid-poor rhodopsin has a time constant of ~ 14 ns and that of lipid, ~4 ns. These time constants do not change with pH or upon illumination. About 30 molecules of Ans are bound per molecule of rhodopsin, and the calculated dissociation constant for binding is 3.2 X -6 M. Measurements of energy transfer from tryptophan residues to bound Ans molecules show no differences in either transfer efficiency or distances between them when rhodopsin is bleached. However, these values vary with pH, indicating that the nature of the light-induced conformational change is different from that of the pH-induced change in the secondary structure of the rhodopsin protein (revealed in the circular dichroism and fluorescence measurements). Temperature dependence of Ans fluorescence intensity shows two reversible thermal transitions in ROS membranes, one at ~7 °C and the other at ~36 °C. These phenomena have been attributed to the phase transitions of the hpid at the two temperatures. The fluorescence of bound Ans is enhanced by lowering the pH and by the presence of Na+ or Ca2+ in the order Ca2+ ≥ Na+. The pronounced effect of Ca2+ cannot be correlated with changes in ionic strength but suggests a specific effect of the divalent ion on membrane conformation.