The Journal of Membrane Biology

, Volume 76, Issue 1, pp 73–82 | Cite as

Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

  • Werner Schmidt


This paper is a continuation of a series of physicochemical studies of vesicle-bound flavins. In this study a detailed analysis of the Sepharose 4B elution profile of various sonicated phospholipids is given which demonstrates the presence of three distinct particle populations. Electron microscopy reveals heterogeneous, multilamellar lipoid aggregates of a diameter of more than 500 Å in fraction I, unilamellar, closed vesicles of 200 to 250 Å diameter in fraction II, and previously unreported micellar structures of approximately 50 Å diameter in fraction III. Fraction II represents vesicles very similar to those obtained by the “deoxycholate procedure.” Fine structure analysis of corrected fluorescence and fluorescence polarization spectra of amphiphilic flavin (AFl 3) bound to vesicles prepared from various phospholipids demonstrates the specificity of the flavin/membrane interaction. When bound to fractions I, II and III of sonicated egg lecithin, AFl 3 exhibits different temperature (i. e. phase-) dependencies. This demonstrates that the flavin/membrane interaction depends strongly upon the particular lipidstructure as well. Evidence is presented which defines the type of excited state of flavin involved in photochemical and photophysical reactions (triplet or singlet). Finally, the dependencies of the photoreactions of vesicle-associated flavins on parameters such as pH, temperature and ionic strength are discussed. Comparison of photoreactions of isotropic and anisotropic flavins also reveal clues for the various mechanisms involved.

Key Words

flavin fluorescence photoreduction singlet state triplet state photo-pK lipid-phase transition 


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  1. 1.
    Biegel, C.M., Gould, J.M. 1982. Kinetics of hydrogen diffusion across phospholipid vesicle membranes.Biochemistry 20:3473–3479Google Scholar
  2. 2.
    Bruice, R.C. 1976. Models and flavin catalysis.In: Progress in Bioanorganic Chemistry. E.T. Kaiser and F.J. Kezdy, editors. Vol. 4, pp. 1–87. John Wiley & Sons, New York-London-TorontoGoogle Scholar
  3. 3.
    Brunner, J., Skrabal, P., Hauser, H., 1976. Single bilayer vesicles prepared without sonication, physico-chemical properties.Biochim. Biophys. Acta 455:322–331Google Scholar
  4. 4.
    Cafiso, D.S., Hubbel, W.L. 1978. Estimation of transmembrane potentials from phase equilibria of hydrophobic paramagnetic ions.Biochemistry 17:187–195Google Scholar
  5. 5.
    Clement, N.R., Gould, J.M. 1980. Viscosity of the internal aqueous phase of unilamellar phospholipid vesicles.Arch. Biochem. Biophys. 202:650–652Google Scholar
  6. 6.
    Clement, N.R., Gould, J.M. 1980. Pyranine (-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles.Biochemistry 20:1534–1538Google Scholar
  7. 7.
    Clement, N.R., Gould, J.M. 1981. Modulation by small hydrophobic molecules of valinomycin-mediated potassium transport across phospholipid vesicle membranes.Biochemistry 20:1539–1543Google Scholar
  8. 8.
    Clement, N.R., Gould, J.M. 1981. Kinetics for development of gramicidin-induced ion permea bility in unilamellar phospholipid vesicles.Biochemistry 20:1544–1548Google Scholar
  9. 9.
    Gutami, A., Hurt, E., Hauska, G. 1979. Vectorial redox reactions of physiological quinones. I. Requirement of a minimum length of isoprenoid side chain.Biochim. Biophys. Acta 547:583–596Google Scholar
  10. 10.
    Gillies, R.J., Deamer, D.W. 1979. Intracellular pH: Methods and application.Curr. Top. Bioenerg. 9:63–87Google Scholar
  11. 11.
    Gutknecht, J., Walter, A. 1981. Hydroxyl ion permeability of lipid bilayer membranes.Biochim. Biophys. Acta 645:161–162Google Scholar
  12. 12.
    Haas, W. 1973. Beitrag zur Flavinabhängigen Photodecarboxylierung und Photodehydrierung. Ph. D. Thesis, Universität KonstanzGoogle Scholar
  13. 13.
    Haas, W., Hemmerich, P. 1979. Flavin-dependent substrate photooxidation as a chemical model of hydrogenase action.Biochem. J. 181:95–105Google Scholar
  14. 14.
    Helenius, A., Simon, K. 1975. Solubilization of membranes by detergents.Biochim. Biophys. Acta 415:29–79Google Scholar
  15. 15.
    Hemmerich, P. 1976. The present status of flavin and flavoenzyme chemistry.Prog. Chem. Org. Natural Prod. 33:451–527Google Scholar
  16. 16.
    Holten, D., Windsor, M.W., Parson, W.W., Gouterman, M. 1978. Models of bacterial photosynthesis: Electron transfer from photoexcited singlet bacteriopheophytin to methyl viologen andm-dinitrobenzene.Photochem. Photobiol. 28:951–961Google Scholar
  17. 17.
    Huang, C. 1969. Studies on phosphatidylcholine vesicles: Formation and physical characteristics.Biochemistry 8:344–352Google Scholar
  18. 18.
    Kano, K., Fendler, J.H. 1978. Pyranine as a sensitive pH probe for liposome interiors and surfaces.Biochim. Biophys. Acta 509:289–299Google Scholar
  19. 19.
    Katagi, T., Yamamura, T., Saito, T., Sasaki, Y. 1981. Electron transport across lipid membranes photosensitized by an amphiphilic zinc porphyrinChem. Lett. pp. 503–506 (Chemical Society of Japan)Google Scholar
  20. 20.
    Knappe, W.-R. 1974. Photochemie des 10 phenylisoalloxazins: Intramolekulare Singlett- und intermolekulare Triplett-Reaktionen.Chem. Ber. 107:1614–1636Google Scholar
  21. 21.
    Lawaczek, R., Blackman, R., Kainosho, M. 1977. Ion permeation across the bilayer of annealed phosphatidylcholine vesicles at elevated temperatures. Concentration dependence and the micelle-bilayer dynamic equilibrium.Biochim. Biophys. Acta 468:411–421Google Scholar
  22. 22.
    Lawaczek, R., Kainosho, M., Giradet, J.-L., Chan, S.I. 1975. Effects of structural defects in sonicated phospholipid vesicles on fusion and ion permeability.Nature (London) 256:584–586Google Scholar
  23. 23.
    Marrink, J., Gruber, M. 1969. Molecular weight determination by chromatography on sephrose 4B.FEBS Lett.4:242–244Google Scholar
  24. 24.
    Michel, H., Hemmerich, P. 1980. Substitution of the flavin chromophore with lipophilic sidechains: A novel membrane redox label.J. Membrane Biol. 60:143–153Google Scholar
  25. 25.
    Michel, H., Schmidt, W., Hemmerich, P. 1982. On the environment and the rotational motion of amphiphilic flavins in artificial membrane vesicles as studied by electron paramagnetic resonance spectroscopy.Biophys. Chem. 15:121–130Google Scholar
  26. 26.
    Mimms, L.T., Zampighi, G., Nozaki, Y., Tanford, C. Reynolds, J.A. 1981 Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside.Biochemistry 20:833–840Google Scholar
  27. 27.
    Nichols, J.W., Deamer, D.W. 1980. Net proton-hydroxyl permeability of large unilamellar liposomes measured by an acid-based titration technique.Proc. Natl. Acad. Sci. USA 77:2038–2042Google Scholar
  28. 28.
    Nozaki, Y., Tanford, C. 1981. Proton and hydroxide ion permeability of phospholipid vesicles.Proc. Natl. Acad. Sci. USA 77:4324–4328Google Scholar
  29. 29.
    Owen, S.D., O'Boyle, A.A. 1971. Photochemical reactions in rigid media-I. The effect of gelation on the aerobic photochemistry of riboflavin in aqueous solution.Photochem. Photobiol. 14:683–692Google Scholar
  30. 30.
    Pohl, W.G. 1982. Kinetics of proton-hydroxyl transport across lecithin vesicle membranes as measured with a lipoid pH-indicator.Z. Naturforsch. 37c:120–128Google Scholar
  31. 31.
    Pohl, W.G., Kreikenbohm, R., Seuwen, K. 1980. The specificity of ionophore A23187 in cation transport across lipid membranes.Z. Naturforsch. 35c:562–568Google Scholar
  32. 32.
    Razin, S. 1972. Reconstitution of biological membranes.Biochim. Biophys. Acta 265:241–296Google Scholar
  33. 33.
    Schmidt, W. 1979. On the environment and the rotational motion of amphiphilic flavins in artificial membrane vesicles as studied by fluorescence.J. Membrane Biol. 47:1–25Google Scholar
  34. 34.
    Schmidt, W. 1980. Artificial flavin/membrane systems: A possible model for physiological bluelight action.In: The Bluelight Syndrome. H. Senger, editor. pp. 212–220. Springer-Verlag BerlinGoogle Scholar
  35. 35.
    Schmidt, W. 1980. A high-performance dual wavelength spectrophotometer and fluorometer.J. Biochem. Biophys. Methods 2:171–181Google Scholar
  36. 36.
    Schmidt, W. 1981. Reply to: Temperature effects on the polarity and localization of amphiphilic flavins in artificial membrane vesicles.J. Membrane Biol. 60:164–165Google Scholar
  37. 37.
    Schmidt, W. 1981. Fluorescence properties of isotropically and anisotropically embedded flavins.Photochem. Photobiol. 34:7–16Google Scholar
  38. 38.
    Schmidt, W. 1982. Light-induced redox cycles of flavins in various alcohol/acetic mixtures.Photochem. Photobiol. 36:699–703Google Scholar
  39. 39.
    Schmidt, W. 1982. A computerized single-beam spectrophotometer: An easy set up.Anal. Biochem. 125:162–167Google Scholar
  40. 40.
    Schmidt, W., Butler, W.L. 1976. Flavin-mediated photoreactions in artificial systems: A possible model for the blue light photoreceptor pigment in living systems.Photochem. Photobiol. 24:71–75Google Scholar
  41. 41.
    Schmidt, W., Hemmerich, P. 1981. On the redoxreactions and accessibility of amphiphilic flavins in artificial membrane vesicles.J. Membrane Biol. 60:129–141Google Scholar
  42. 42.
    Schreiner, S., Kramer, H.E.A. 1976. Influence of pH on flavins in the triplet state.In: Flavins and Flavoproteins. T.P. Singer, editor. pp. 793–799. Elsevier Scieince, AmsterdamGoogle Scholar
  43. 43.
    Schreiner, S., Steiner, U., Kramer, H.E.A. 1975. Determination of the pK values of the lumiflavin triplet by flash photolysis.Photochem. Photobiol. 21:81–84Google Scholar
  44. 44.
    Song, P.-S., Metzler, D.E. 1967. Photochemical degradation of flavins. IV. Studies of the Anaerobic Photolysis of Riboflavin.Photochem. Photobiol. 6:691–709Google Scholar
  45. 45.
    Turro, N.J., Lehr, G.F., Butcher, J.A., Jr. 1982. Temperature dependence of the cycloaddition of phenylchlorocarbene to alkenes. Observation of “Negative Activation Energies.”J. Am. Chem. Soc. 104:1754–1756Google Scholar
  46. 46.
    Van der Bosch, J., McConnell, H.M. 1975. Fusion of dipalmitoylphosphatidylcholine vesicle membranes induced by concanavalin A.Proc. Natl. Acad. Sci. USA 72:4409–4413Google Scholar

Copyright information

© Springer-Verlag 1983

Authors and Affiliations

  • Werner Schmidt
    • 1
  1. 1.Fakultät für BiologieUniversität KonstanzKonstanzW. Germany

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