The Journal of Membrane Biology

, Volume 33, Issue 1, pp 63–108 | Cite as

1-Anilino-8-naphthalenesulfonate: A fluorescent probe of ion and ionophore transport kinetics and trans-membrane asymmetry

  • Duncan H. Haynes
  • Philip Simkowitz


The kinetics of the transport of the 1-anilino-8-naphthalenesulfonate (ANS, an anionic fluorescent probe of the membrane surface) across phospholipid vesicle membranes have been studied using a stopped-flow rapid kinetic technique. The method has been used to gain detailed information about the mechanism of transport of this probe and to study ionophore-mediated cation transport across the membrane. The technique has also been exploited to study differences between the inside and outside surfaces of vesicles containing phosphatidyl choline (PC).

The following is a summary of the major conclusions of this study. (a) Binding of ANS on the outside surface occurs within times shorter than 100 μsec while permeation occurs in the time range 5–100 sec. (b) Net transport of ANS occurs with cotransport of alkali cations. (c) The transport rate is maximal in the region of the crystalline to liquidcrystalline phase transition, and the increase correlates with changes in the degree of aggregation of the vesicles. (d) Incorporation of phosphatidic acid (PA), phosphatidyl ethanolamine (PE) or cholesterol into PC membranes decreases the rate of ANS transport. (e) Neutral ionophores (I) of the valinomycin type increase ANS permeability in the presence of alkali cations (M+) by a mechanism involving the transport of a ternaryI−M+-ANS complex. The equilibrium constants for formation of these complexes and their rate constants for their permeation are presented. The maximal turnover number for ANS transport by valinomycin in dimyristoyl PC vesicles at 35°C was 46 per sec. (f) The partitioning of the ionophore between the aqueous and membrane phases and the rate of transfer of an ionophore from one membrane have been determined in kinetic experiments. (g) A method is described for the detection ofI−M+ complexes on the membrane surface by their enhancement effects on ANS fluorescence at temperature below the phase transition temperature on “monolayer” vesicles. The apparent stability constants for severalI−M+ complexes are given. (h) Analysis of the effect of ionic strength on the ANS binding to the inside outside surfaces indicates that the electrostatic surface potential (at fixed ionic strength and surface change) is larger for the inside surface than for the outside surface. (i) Analysis of the dependence of the maximal ANS binding for the inside and outside surfaces of vesicles made from PC and a variable mole fraction of PA, PE or cholesterol indicate that the latter three are located preferentially on the inside surface.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Azzi, A., Chance, B., Radda, G.K., Lee, C.P. 1969. A fluorescence probe of energydependent structure changes in fragmented membranes.Proc. Nat. Acad. Sci. USA 62:612Google Scholar
  2. Bakker, E.P., Dam, K. van 1974. Influence of diffusion potentials across liposomal membranes on the fluorescence intensity of 1-anilino-8-naphthalenesulfonate.Biochim. Biophys. Acta 339:157Google Scholar
  3. Benz, R., Stark, G., Janko, K., Läuger, P. 1973. Valinomycin-mediated ion transport through neutral lipid membranes: Influence of hydrocarbon chain length and temperature.J. Membrane Biol. 14:339Google Scholar
  4. Bessette, F., Seufert, W.D. 1975. Increase in fluorescence energy transfer across lipid bilayers induced by valinomycin.Biochim. Biophys. Acta 373(1):10Google Scholar
  5. Blok, M.C., Gier, J. de, Van Deenen, L.L.M. 1974. Kinetics of the valinomycin-induced potassium ion leak from liposomes with potassium thiocyanate enclosed.Biochim. Biophys. Acta 367:210Google Scholar
  6. Chapman, D. 1965. The Structure of Lipids. Methuen, LondonGoogle Scholar
  7. Chapman, D., Byrne, P., Shipley, G.G. 1966. Physical studies of phospholipids. I. Solid state and mesomorphic properties of some 2,3-diacyl-Dl-phospatidylethanolamines.Proc. R. Soc. London A 290:115Google Scholar
  8. Chapman, D., Williams, R.M., Ladbrooke, B.D. 1967. Physical studies of phospholipids. VI. Thermotropic and lyotropic mesomorphism of some 1,2-diacylphosphatidylcholines (lecithins).Chem. Phys. Lipids 1:445Google Scholar
  9. Ciani, S., Eisenman, G., Szabo, G. 1969. A theory for the effects of neutral carriers such as the macrotetralide actin antibiotics on the electric properties of bilayer membranes.J. Membrane Biol. 1:1Google Scholar
  10. Conti, F., Tasaki, I., Wanke, E. 1971. Fluorescence signals in ANS-stained squid giant axons during voltage-clamp.Biophysik 8:58Google Scholar
  11. Cornelius, G., Gärtner, W., Haynes, D.H. 1974. Cation complexion by valinomycin-and nigericin-type ionophores registered by the fluorescence signal of Tl+.Biochemistry 13:3052Google Scholar
  12. Davis, D.G., Tosteson, D.C. 1971. Interaction between valinomycin and K+, Na+, and anions in CDCl3 and hexane.Biophys. J. 11:310a Google Scholar
  13. Davis, D.G., Tosteson, D.C. 1975. Nuclear magnetic resonance studies of the interactions of anions and solvent with cation complexes of valinomycin.Biochemistry 14:3962Google Scholar
  14. Devaux, P., McConnell, H.M. 1972. Lateral diffusion in spin-labelled phosphatidylcholine multilayers.J. Am. Chem. Soc. 94:4475Google Scholar
  15. Eisenman, G., Ciani, S., Szabo, G. 1969. The effects of the macrotetralide actin antibiotics on the equilibrium extraction of alkali metal salts into organic solvents.J. Membrane Biol. 1:294Google Scholar
  16. Gains, N., Dawson, A.P. 1975. Transmembrane electrophoresis of 8-anilino-1-naphthalenesulfonate through egg lecithin liposome membranes.J. Membrane Biol. 24:237Google Scholar
  17. Gier, J. de, Mandersloot, J.G., Deenen, L.L.M. van. 1968. Lipid composition and permeability of liposomes.Biochim. Biophys. Acta 150:666Google Scholar
  18. Haynes, D.H. 1972. Detection of ionophore-cation complexes on phospholipid membranes.Biochim. Biophys. Acta 255:406Google Scholar
  19. Haynes, D.H. 1974. 1-anilino-8-naphthalenesulfonate: A fluorescent indicator of ion binding and electrostatic potential on the membrane surface.J. Membrane Biol. 17:341Google Scholar
  20. Haynes, D.H. 1977. Metal-ligand interactions in organic and biochemistry.In: 9th Jerusalem Symposium. B. Pullman, editor. J. Reidel Publishing, Dordrecht-Holland (in press)Google Scholar
  21. Haynes, D.H., Pressman, B.C. 1974. Two-phase partition studies of alkali cation complexation by ionophores.J. Membrane Biol. 18:1Google Scholar
  22. Haynes, D.H., Pressman, B.C., Kowalsky, A. 1971. A nuclear magnetic resonance study of23Na+ complexing by ionophores.Biochem. 10:852Google Scholar
  23. Haynes, D.H., Staerk, H. 1974. 1-anilino-8-naphthalenesulfonate: A fluorescent probe of membrane surface structure, composition and mobility.J. Membrane Biol. 17:313Google Scholar
  24. Haynes, D.H., Wiens, T., Pressman, B.C. 1974. Turnover numbers for ionophorecatalyzed cation transport across the mitochondrial membrane.J. Membrane Biol. 18:23Google Scholar
  25. Huang, C. 1969. Studies on phosphatidylcholine vesicles. Formation and physical characteristics.Biochemistry 8:344Google Scholar
  26. Huang, C.H., Sipe, J.P., Chow, S.T., Martin, R.B. 1974. Differential interaction of cholesterol with phosphatidylcholine on the inner and outer surface of lipid bilayer vesicles.Proc. Nat. Acad. Sci. USA 71:359Google Scholar
  27. Israelachvili, J.M. 1973. Theoretical considerations on the asymmetric distribution of charged phospholipid molecules on the inner and outer layers of curved bilayer membranes.Biochim. Biophys. Acta 323:659Google Scholar
  28. Jacobson, K., Papahadjopoulos, D. 1975. Phase transition and phase separation in phospholipid membranes induced by changes in temperature, pH, and concentration in bivalent cations.J. Biochem. 14(1):152Google Scholar
  29. Jacobson, K., Papahadjopoulos, D. 1976. Effect of a phase transition on the binding of 1-anilino-8-naphthalenesulfonate to phospholipid membranes.Biophys. J. 16:549Google Scholar
  30. Johnson, S.M. 1973. The effect of charge and cholesterol on the size and thickness of sonicated phospholipid vesicles.Biochim. Biophys. Acta 307:27Google Scholar
  31. Johnson, S.M., Bangham, A.D. 1969. Potassium permeability of single compartment liposomes with and without valinomycin.Biochim. Biophys. Acta 193(1):82Google Scholar
  32. Ketterer, B., Neumcke, B., Läuger, P. 1971. Transport mechanism of hydrophobic ions through lipid bilayer membranes.J. Membrane Biol. 5:225Google Scholar
  33. Ladbrooke, B.D., Chapman, D. 1969. Thermal analysis of lipids, proteins and biological membranes. Review and summary of some recent studies.Chem. Phys. Lipids 3(4):304Google Scholar
  34. Lansman, J., Haynes, D.H. 1975. Kinetics of Ca++-triggered membrane aggregation reaction of phospholipid membranes.Biochim. Biophys. Acta. 394:335Google Scholar
  35. Litman, B.J. 1973. Lipid model membranes: Characterization of mixed phospholipid vesicles.Biochemistry 12:2545Google Scholar
  36. Loeb, A.L., Overbeek, J.T.G., Wiersema, P.H. 1961. The electrical double layer around a spherical colloid particle. p. 19. M.I.T. Press, CambridgeGoogle Scholar
  37. Marinetti, G.V., Love, R. 1974. Extent of cross-linking of amino-phospholipids neighbours in the erythrocyte membrane as influenced by the concentration of difluorodinitrobenzene.Biochem. Biophys. Res. Commun. 61:30Google Scholar
  38. McLaughlin, S.G.A., Szabo, G., Ciani, S., Eisenman, G. 1972. The effects of a cyclic polyether on the electrical properties of phospholipid bilayer membranes.J. Membrane Biol. 9:3Google Scholar
  39. Michaelson, D.M., Horwitz, A.F., Klein, M.P. 1973. Transbilayer asymmetry and surface homogeneity of mixed phospholipid in cosonicated vesicles.Biochemistry 12:2637Google Scholar
  40. Papahadjopoulos, D., Miller, N. 1967. Phospholipid model membranes. I. Structural characteristics of hydrated liquid crystals.Biochim. Biophys. Acta 135:624Google Scholar
  41. Papahadjopoulos, D., Poste, G., Schaeffer, B.E., Bail, W.J. 1974. Membrane fusion and molecular segregation in phospholipid vesicles.Biochim. Biophys. Acta 352:10Google Scholar
  42. Papahadjopoulos, D., Watkins, J.C. 1969. Phospholipid model membranes. II. Permeability properties of hydrated liquid crystals.Biochim. Biophys. Acta 135:639Google Scholar
  43. Parsegian, A. 1969. Energy of an ion crossing a low dielectric membrane: Solutions to four relevant electrostatic problems.Nature (London) 221:844Google Scholar
  44. Phillips, M.C., Williams, R.M., Chapman, D. 1969. On the nature of hydrocarbon chain motions in lipid liquid crystals.Chem. Phys. Lipids 3:234Google Scholar
  45. Sims, P.J., Waggoner, A.S., Wang, C.H., Hoffman, J.F. 1974. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles.Biochemistry 13:3315Google Scholar
  46. Stark, G., Benz, R. 1971. The transport of potassium through lipid bilayer membranes by the neutral carriers valinomycin and monactin. Experimental studies to a previously proposed model.J. Membrane Biol. 5:133Google Scholar
  47. Stark, G., Ketterer, B., Benz, R., Läuger, P. 1971. The rate constants of valinomycinmediated ion transport through thin lipid membranes.Biophys. J. 11:981Google Scholar
  48. Szabo, G., Eisenman, G., Ciani, S. 1969. The effects of the macrotetralide actin antibiotics on the electrical properties of phospholipid bilayer membranes.J. Membrane Biol. 1:346Google Scholar
  49. Träuble, H., Grell, E. 1971. The formation of asymmetrical spherical lecithin vesicles.Neurosci. Res. Prog. Bull. 9(3):373Google Scholar
  50. Träuble, H., Haynes, D.H. 1971. The volume change in lipid bilayer lamellae at the crystalline-liquid crystalline phase transition.Chem. Phys. Lipids 7:324Google Scholar
  51. Träuble, H., Sackmann, E. 1972. Studies of the crystalline-liquid crystalline phase transition of lipid model membranes. III. Structure of a steroid-lecithin system below and above the lipid-phase transition.J. Am. Chem. Soc. 94:4499Google Scholar
  52. Tsong, T.Y. 1975a. Effect of phase transition on the kinetics of dye transport in phospholipid bilayer structures.Biochemistry 14:5409Google Scholar
  53. Tsong, T.Y. 1975b. Transport of 1-anilino-8-naphthalenesulfonate as a probe of the effect of cholesterol on the phospholipid bilayer structures.Biochemistry 13:5415Google Scholar
  54. Vanderkooi, J., Martonosi, A. 1971a. Sarcoplasmic reticulum. XII: The interaction of 8-anilino-1-naphthalenesulfonate with skeletal muscle microsomes.Arch. Biochem. Biophys. 144:87Google Scholar
  55. Vanderkooi, J.M., Martonosi, A. 1971b. Sarcoplasmic reticulum. XIII. Changes in the fluorescence of 8-anilino-1-naphthalenesulfonate during Ca2+ transport.Arch. Biochem. Biophys. 144:99Google Scholar
  56. Verkleji, A.J., Zwaal, R.F.A., Roelofsen, B., Comfeurius, P., Kastelijn, D., Deenen, L.L.M. van. 1973. The asymmetric distribution of phospholipids in the human red cell membrane. A combination study using phospholipases and freeze-etch electron microscopy.Biochim. Biophys. Acta 323:178Google Scholar
  57. Yi, P.N., MacDonald, R.C. 1973. Temperature-dependence of optical properties of aqueous dispersions of phosphatidylcholine.Chem. Phys. Lipids 11:114Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1977

Authors and Affiliations

  • Duncan H. Haynes
    • 1
  • Philip Simkowitz
    • 1
  1. 1.Department of PharmacologyUniversity of Miami Medical SchoolMiami

Personalised recommendations