European Biophysics Journal

, Volume 17, Issue 1, pp 25–36

Fluorescence quenching with lindane in small unilamellar l,α-dimyristoylphosphatidylcholine vesicles

  • D. Daems
  • N. Boens
  • F. C. De Schryver


The lateral mobility and lipid-water partition of the pesticide lindane was studied by fluorescence quenching of N-isopropylcarbazole (NIPC) and l,α-palmitoyl-β-(N-carbazolyl) undecanoylphosphatidylcholine (PCUPC) in liposomes of dimyristoylphosphatidylcholine at 50°C. In isotropic solvents the quenching reaction was highly inefficient. A scheme for dynamic quenching, in which the monomolecular quenching rate constant is small, was valid. In lipid bilayers the same scheme was applied to describe the quenching results but the rate constant of the backreaction of the excited complex to quencher and excited probe was of comparable magnitude to the monomolecular quenching rate constant. This phenomenon results in biexponential decays of the fluorescent probe in the presence of quencher. All the rate constants of the scheme could be determined. Stern-Volmer plots at different membrane concentrations were obtained from fluorescence intensity and decay time measurements. From these plots the true bimolecular quenching rate constant, kq, and the rate constant for lateral diffusion, kd, were determined: \(k_{q[NIPC]} = 3.2 \pm 0.5x10^8 M^{ - 1} s^{ - 1} ,k_{q[PCUPC]} = 1.9 \pm 0.4x10^8 M^{ - 1} s^{ - 1} ,k_{d[NIPC]} = 6.6 \pm 0.8x10^8 M^{ - 1} s^{ - 1} \). The smaller value of kq compared to kd for the quenching reaction of NIPC with lindane indicates that this quenching reaction is not diffusion controlled. The lateral diffusion coefficient D of lindane was found to be 1.7±0.2×10-6 cm2/s in dimyristoylphosphatidylcholine vesicles at 50°C. The partition coefficient of lindane in these lipid bilayers is very high (>2000).

Key words

Diffusion coefficient dimyristoylphosphatidylcholine fluorescence quenching lindane partition coefficient 





1,2,3,4,5,6-hexachlorocyclohexane (γ-isomer)




l,α-palmitoyl-β-(N-carbazolyl) undecanoylphosphatidylcholine


small unilamellar vesicles


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  1. Ahmad A, Durocher G (1981) Fluorescence quenching of carbazole by halocarbons in 3-methylpentane solutions at room teperature. Can J Spectrosc 26:19–24Google Scholar
  2. Almgren M, Grieser F, Thomas JK (1979) Dynamic and static aspects of solubilization of neutral arenes in ionic micellar solutions. J Am Chem Soc 101:279–291Google Scholar
  3. Antunes-Madeira MC, Madeira VMC (1985) Partition of lindane in synthetic and native membranes. Biochim Biophys Acta 820:165–172Google Scholar
  4. Barenholz Y, Gibbes D, Litman BJ, Goll J, Thompson TE, Carson FD (1977) A simple method for the preparation of homogenous phospholipid vesicles. Biochemistry 16:2806–2810Google Scholar
  5. Birks JB (1970) Photophysics of aromatic molecules. Wiley-Interscience, London, pp 301–371Google Scholar
  6. Blatt E, Sawyer WH (1985) Depth-dependent fluorescent quenching in micelles and membranes. Biochim Biophys Acta 822:43–62Google Scholar
  7. Blatt E, Chatelier RC, Sawyer WH (1984) Partition and binding constants in micelles and vesicles from fluorescence quenching data. Chem Phys Lett 108:397–400Google Scholar
  8. Boens N, Van den Zegel M, De Schryver FC (1984) Picosecond lifetime determination of the second excited singlet state of xanthione in solution. Chem Phys Lett 11:340–346Google Scholar
  9. Boens N, Van den Zegel M, De Schryver FC, Desie G (1987) The time-correlated single photon counting technique as a tool in photobiology. In: Favre A, Tyrrell R, Cadet J (eds) From photophysics to photobiology. Elsevier, Amsterdam, pp 93–108Google Scholar
  10. Boens N, Ameloot M, Yamazaki I, De Schryver FC (1988) On the use and the performance of the delta function convolution method for the estimation of fluorescence decay parameters. Chem Phys 121:73–86Google Scholar
  11. Daems D, Boens N, De Schryver FC (1988) Determination of partition and diffusion of lindane in unilamellar L,α-dimyristoylphosphatidylcholine vesicles by fluorescence quenching of the intramembrane probe methyl 11-(N-carbazolyl) undecanoate. Anal Chim Acta 205:61–75Google Scholar
  12. Deumié M, Viallet P, Lightcap D, Rhyne RH Jr, Wade CG (1981) Mecanismes spécifiques d'inhibition de la fluorescence dans les membranes d'érythrocytes: étude du pyrène et du benzo (a) pyréne par des inhibiteurs liposolubles. J Chim Phys 78:475–483Google Scholar
  13. Dittmer JC, Lester RL (1964) A simple, specific spray for the detection of phospholipids on thin-layer chromatograms. J Lipid Res. 5:126–127Google Scholar
  14. Eigen M (1960) Relaxationsspektren chemischer Umwandlungen (Metallkomplexe und protolytische Reaktionen in wässriger Lösung). Z Elektrochem 64:115–123Google Scholar
  15. Encinas MV, Lissi EA (1982) Evaluation of partition constants in compartmentalised systems from fluorescence quenching data. Chem Phys Lett 91:55–57Google Scholar
  16. Gösele UM (1984) Reaction kinetics and diffusion in condensed matter. Prog React Kinet 13:63–161Google Scholar
  17. Gupta CM, Radhakrishnan R, Khorana HG (1977) Glycerophospholipid synthesis: Improved general method and new analogs containing photoactivable groups. Proc Natl Acad Sci USA 74:4315–4319Google Scholar
  18. Huang C (1969) Studies on phosphatidylcholine vesicles. Formation and physical characteristics. Biochemistry 8: 344–352Google Scholar
  19. Kano K, Kawazumi H, Ogawa T, Sunamoto J (1981) Fluorescence quenching in liposomal membranes. Exciplex as a probe for investigating artificial lipid membrane properties. J Phys Chem 85:2204–2209Google Scholar
  20. Lakowicz JR (1983) Principles of fluorescence spectroscopy. Plenum Press, New York, pp 257–302Google Scholar
  21. Lakowicz JR, Hogen D (1980) Chlorinated hydrocarbon-cell membrane interactions studied by the fluorescence quenching of carbazole-labeled phospholipids: probe synthesis and characterization of the quenching methodology. Chem Phys Lipids 26:1–40Google Scholar
  22. Lakowicz JR, Hogen D, Omann G (1977) Diffusion and partitioning of a pesticide, lindane, into phosphatidylcholine bilayers. A new fluorescence quenching method to study chlorinated hydrocarbon-membrane interactions. Biochim Biophys Acta 471:401–411Google Scholar
  23. Marquardt DW (1963) An algorithm for least-squares estimation of nonlinear parameters. J Soc Ind Appl Math 11:431–441Google Scholar
  24. McCracken MS, Holt NJ (1985) High-performance liquid chromatographic determination of lipids in vesicles. J Chromatogr 348:221–227Google Scholar
  25. O'Connor DV, Phillips D (1984) Time-correlated single photon counting. Academic Press, LondonGoogle Scholar
  26. Omann GM, Glaser M (1984) Biosynthetic incorporation of fluorescent carbazolylundecanoic acid into membrane phospholipids of LM cells and determination of quenching constants and partition coefficients of hydrophobic quenchers. Biochemistry 23:4962–4969Google Scholar
  27. Omann GM, Lakowicz JR (1982) Interactions of chlorinated hydrocarbon insecticides with membranes. Biochim Biophys Acta 684:83–95Google Scholar
  28. Owen CS (1975) Two dimensional diffusion theory: cylindrical diffusion model applied to fluorescence quenching. J Chem Phys 62:3204–3207Google Scholar
  29. Scatchard G (1949) The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51:660–672Google Scholar
  30. Selinger Z, Lapidot Y (1966) Synthesis of fatty acid anyhydrides by reaction with dicylohexylcarbodiimide. J Lipid Res 7: 174–175Google Scholar
  31. Sikaris KA, Thulborn KR, Sawyer WH (1981) Resolution of partition coefficients in the transverse plane of the lipid bilayer. Chem Phys Lipids 29:23–36Google Scholar
  32. Smoluchowski M von (1917) Versuch einer mathematischen Theorie der Koagulationskinetik koloider Lösunger. Z Phys Chem 92:129–168Google Scholar
  33. Stewart JCM (1980) Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal Biochem 104:10–14Google Scholar
  34. Vandendriessche J, Palmans P, Toppet S, Boens N, De Schryver FC, Masuhara H (1984) Configurational and conformational aspects in the excimer formation of bis (carbazoles). J Am Chem Soc 106:8057–8064Google Scholar
  35. Van den Zegel M, Boens N, De Schryver FC (1984) Fluorescence decay of 1-methylpyrene in small unilamellar dimyristoylphosphatidylcholine vesicles. A temperature and concentration dependent study. Biophys Chem 20:333–345Google Scholar
  36. Van den Zegel M, Boens N, Daems D, De Schryver FC (1986) Possibilities and limitations of the time-correlated single photon counting technique: A comparative study of correction methods for the wavelength dependence of the instrument response function. Chem Phys 101:311–335Google Scholar
  37. Waka Y, Mataga N, Tanaka F (1980) Behavior of heteroexcimer systems in single bilayer liposomes. Photochem Photobiol 32:335–340Google Scholar
  38. Washington K, Sarasua MM, Koehler LS, Koehler KA, Schultz JA, Pedersen LG, Hiskey RG (1984) Utilization of heavyatom effect quenching of pyrene fluorescence to determine the intramembrane distribution of halothane. Photochem Photobiol 40:693–701Google Scholar
  39. Zuker M, Szabo AG, Bramhall L, Krajcarski DT, Selinger B (1985) Delta function convolution method (DFCM) for fluorescence decay experiments. Rev Sci Instrum 56:14–22Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • D. Daems
    • 1
  • N. Boens
    • 1
  • F. C. De Schryver
    • 1
  1. 1.Department of ChemistryKatholieke Universiteit LeuvenHeverleeBelgium

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