INTERACTION OF FLUORESCENT PROBES WITH MEMBRANES .I. EFFECT OF IONS ON ERYTHROCYTE MEMBRANES

1-Anilinonaphthalene-8-sulfonate, a fluorescent probe, does not fluoresce in water but fluoresces strongly in organic solvents and when bound to certain native proteins. The presented studies indicate that 1-anilinonaphthalene-8- sulfonate binds with enhanced fluorescence to hemoglobinfree rabbit erythrocyte membranes. This binding is sensitive to the cation concentration of the suspending medium. Thus 1- anilinonaphthalene-8-sulfonate binds to hemoglobin-free erythrocyte membranes in 20 mosm Tris-HCl buffer (pH 7.4) to the extent of 1.05 × 107 molecules/cell with a statistical binding constant, Kapp of 4.3 × 10-5 M. Addition of 300 mosm NaCl or 3.0 mosm CaCl2 increases the number of 1-anilinonaphthalene- 8-sulfonate molecules bound to 3.14 × 107 and 2.83 × 107 molecules per cell, respectively, while the Kapp values decrease to 3.45 and 2.70 × 10-5 M, respectively. Addition of NaCl to hemoglobin-free erythrocyte membranes suspended in hypotonic 20 mosm Tris-HCl buffer (pH 7.4) leads to volume changes of the membranes which indicate an osmotic response and thus the restoration of the membrane's impermeability to cations. An attempt is made to corrolate these volume changes with the effect of the ions on the membrane per se which bring about the enhanced binding of 1- anilinonaphthalene-8-sulfonate. Results indicate that the addition of low concentrations of cations leads to shielding by the ion atmosphere, of the electrostatic repulsion of ionic groups of membrane phospholipids which could explain the enhanced binding of 1-anilinonaphthalene-8-sulfonate and the restoration of cation impermeability. Studies on the binding of 1- anilinonaphthalene-8-sulfonate to detergent micelles indicate that 1-anilinonaphthalene-8-sulfonate is a probe for hydrophobic- hydrophylic interphases containing neutral or cationic hydrophilic groups. © 1969, American Chemical Society. All rights reserved.