Skip to main content
SpringerLink
Log in

The New Fluorescent Membrane Probe Ahba: A Comparative Study with the Largely Used Laurdan

  • ORIGINAL PAPER
  • Published:
Journal of Fluorescence Aims and scope Submit manuscript

Abstract

Lipid bilayers have been largely used as model systems for biological membranes. Hence, their structures, and alterations caused on them by biological active molecules, have been the subject of many studies. Accordingly, fluorescent probes incorporated into lipid bilayers have been extensively used for characterizing lipid bilayer fluidity and/or polarity. However, for the proper analysis of the alterations undergone by a membrane, a comprehensive knowledge of the fluorescent properties of the probe is fundamental. Therefore, the present work compares fluorescent properties of a relative new fluorescent membrane probe, 2-amino-N-hexadecyl-benzamide (Ahba), with the largely used probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), using both static and time resolved fluorescence. Both Ahba and Laurdan have the fluorescent moiety close to the bilayer surface; Ahba has a rather small fluorescent moiety, which was shown to be very sensitive to the bilayer surface pH. The main goal was to point out the fluorescent properties of each probe that are most sensitive to structural alterations on a lipid bilayer. The two probes were incorporated into bilayers of the well-studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which exhibits a gel-fluid transition around 23 °C. The system was monitored between 5 and 50 °C, hence allowing the study of the two different lipid structures, the gel and fluid bilayer phases, and the transition between them. As it is known, the fluorescent emission spectrum of Laurdan is highly sensitive to the bilayer gel-fluid transition, whereas the Ahba fluorescence spectrum was found to be insensitive to changes in bilayer structure and polarity, which are known to happen at the gel-fluid transition. However, both probes monitor the bilayer gel-fluid transition through fluorescence anisotropy measurements. With time-resolved fluorescence, it was possible to show that bilayer structural variations can be monitored by Laurdan excited state lifetimes changes, whereas Ahba lifetimes were found to be insensitive to bilayer structural modifications. Through anisotropy time decay measurements, both probes could monitor structural bilayer changes, but the limiting anisotropy was found to be a better parameter than the rotational correlation time. It is interesting to have in mind that the relatively small fluorophore of Ahba (o-Abz) could possibly be bound to a phospholipid hydrocarbon chain, not disturbing much the bilayer packing and being a sensitive probe for the bilayer core.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Isenberg G, Niggli V, Kwang WJ (1997) Interaction of cytoskeletal proteins with membrane lipids. In: International review of cytology, vol Volume 178. Academic Press, pp 73–125

  2. Epand RM, Kraayenhof R (1999) Fluorescent probes used to monitor membrane interfacial polarity. Chem Phys Lipids 101(1):57–64

    Article  PubMed  CAS  Google Scholar 

  3. Winterhalter M (2000) Black lipid membranes. Curr Opin Colloid Interface Sci 5(3–4):250–255

    Article  CAS  Google Scholar 

  4. Gruszecki WI, Gagos M, Herec M, Kernen P (2003) Organization of antibiotic amphotericin B in model lipid membranes. A mini review. Cell Mol Biol Lett 8(1):161–170

    PubMed  CAS  Google Scholar 

  5. Lindblom G, Gröbner G (2006) NMR on lipid membranes and their proteins. Curr Opin Colloid Interface Sci 11(1):24–29

    Article  CAS  Google Scholar 

  6. Chiang Y, Costa-Filho A, Freed J (2007) Two-dimensional ELDOR in the study of model and biological membranes. Appl Magn Reson 31(3):375–386

    Article  CAS  Google Scholar 

  7. Flanders BN, Dunn RC, Toyoko I (2007) Chapter 5 near-field scanning optical microscopy of lipid membranes. In: Interface science and technology, vol Volume 14. Elsevier, pp 117–140

  8. Demchenko AP, Mely Y, Duportail G, Klymchenko AS (2009) Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes. Biophys J 96(9):3461–3470

    Article  PubMed  CAS  Google Scholar 

  9. Weber G, Farris FJ (1979) Synthesis and spectral properties of a hydrophobic fluorescent-probe - 6-propionyl-2-(dimethylamino)naphthalene. Biochemistry 18(14):3075–3078

    Article  PubMed  CAS  Google Scholar 

  10. Parasassi T, Destasio G, Dubaldo A, Gratton E (1990) Phase fluctuation in phospholipid-membranes revealed by Laurdan Fluorescence. Biophys J 57(6):1179–1186

    Article  PubMed  CAS  Google Scholar 

  11. Parasassi T, Destasio G, Ravagnan G, Rusch R, Gratton E (1991) Quantitation of lipid phases in phospholipid-vesicles by the generalized polarization of Laurdan fluorescence. Biophys J 60(1):179–189

    Article  PubMed  CAS  Google Scholar 

  12. Ferretti G, Taus M, Dousset N, Solera ML, Valdiguie P, Curatola G (1993) physicochemical properties of copper-oxidized high-density-lipoprotein—a fluorescence study. Biochem Mol Biol Int 30(4):713–719

    PubMed  CAS  Google Scholar 

  13. Ambrosini A, Bertoli E, Tanfani F, Wozniak M, Zolese G (1994) The effect of n-acyl ethanolamines on phosphatidylethanolamine phase-transitions studied by Laurdan generalized polarization. Chem Phys Lipids 72(2):127–134

    Article  PubMed  CAS  Google Scholar 

  14. Alleva R, Ferretti G, Borghi B, Pignotti E, Bassi A, Curatola G (1995) Physicochemical properties of membranes of recovered erythrocytes in blood autologous transfusion—a study using fluorescence technique. Transfus Sci 16(3):291–297

    Article  PubMed  CAS  Google Scholar 

  15. Bell JD, Burnside M, Owen JA, Royall ML, Baker ML (1996) Relationships between bilayer structure and phospholipase A(2) activity: interactions among temperature, diacylglycerol, lysolecithin, palmitic acid, and dipalmitoylphosphatidylcholine. Biochemistry 35(15):4945–4955

    Article  PubMed  CAS  Google Scholar 

  16. Bagatolli LA, Maggio B, Aguilar F, Sotomayor CP, Fidelio GD (1997) Laurdan properties in glycosphingolipid-phospholipid mixtures: a comparative fluorescence and calorimetric study. BiochimBiophys Acta Biomembr 1325(1):80–90

    Article  CAS  Google Scholar 

  17. Bagatolli LA, Gratton E, Fidelio GD (1998) Water dynamics in glycosphingolipid aggregates studied by LAURDAN fluorescence. Biophys J 75(1):331–341

    Article  PubMed  CAS  Google Scholar 

  18. Brunelli R, Mei G, Krasnowska EK, Pierucci F, Zichella L, Ursini F, Parasassi T (2000) Estradiol enhances the resistance of LDL to oxidation by stabilizing apoB-100 conformation. Biochemistry 39(45):13897–13903

    Article  PubMed  CAS  Google Scholar 

  19. Ambrosini A, Zolese G, Balercia G, Bertoli E, Arnaldi G, Mantero F (2001) Laurdan* fluorescence: a simple method to evaluate sperm plasma membrane alterations. Fertil Steril 76(3):501–505

    Article  PubMed  CAS  Google Scholar 

  20. Gaus K, Gratton E, Kable EPW, Jones AS, Gelissen I, Kritharides L, Jessup W (2003) Visualizing lipid structure and raft domains in living cells with two-photon microscopy. Proc Natl Acad Sci U S A 100(26):15554–15559

    Article  PubMed  CAS  Google Scholar 

  21. De Vequi-Suplicy CC, Benatti CR, Lamy MT (2006) Laurdan in fluid bilayers: position and structural sensitivity. J Fluoresc 16(3):431–439

    Article  PubMed  CAS  Google Scholar 

  22. Moyano F, Silber JJ, Correa NM (2008) On the investigation of the bilayer functionalities of 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine (DOPC) large unilamellar vesicles using cationic hemicyanines as optical probes: a wavelength-selective fluorescence approach. J Colloid Interface Sci 317(1):332–345

    Article  PubMed  CAS  Google Scholar 

  23. Lucio AD, Vequi-Suplicy CC, Fernandez RM, Lamy MT (2010) Laurdan spectrum decomposition as a tool for the analysis of surface bilayer structure and polarity: a study with DMPG, peptides and cholesterol. J Fluoresc 20(2):473–482

    Article  PubMed  CAS  Google Scholar 

  24. Marquezin CA, Hirata IY, Juliano L, Ito AS (2006) Spectroscopic characterization of 2-amino-N-hexadecyl-benzamide (AHBA), a new fluorescence probe for membranes. Biophys Chem 124(2):125–133

    Article  PubMed  CAS  Google Scholar 

  25. Turchiello RF, Lamy-Freund MT, Hirata IY, Juliano L, Ito AS (1998) Ortho-aminobenzoic acid as a fluorescent probe for the interaction between peptides and micelles. Biophys Chem 73(3):217–225

    Article  PubMed  CAS  Google Scholar 

  26. Ito AS, Takara M (2005) General and specific solvent effects in optical spectra of ortho-aminobenzoic acid. J Fluoresc 15(2):171–177

    Article  PubMed  Google Scholar 

  27. Ito AS, Takara M, Eisenhut JK, Hirata IY, Juliano L (2009) Solvent effects in optical spectra of ortho-aminobenzoic acid derivatives. J Fluoresc 19(6):1053–1060

    Article  PubMed  Google Scholar 

  28. Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Plenum Publishers, New York

    Book  Google Scholar 

  29. Heimburg T (2007) Thermal biophysics of membranes. Tutorials in biophysics. Wiley-VCH, Weinheim

    Book  Google Scholar 

  30. Parasassi T, Conti F, Gratton E (1986) Time-resolved fluorescence emission-spectra of Laurdan in phospholipid-vesicles by multifrequency phase and modulation fluorometry. Cell Mol Biol 32(1):103–108

    PubMed  CAS  Google Scholar 

  31. Rottenberg H (1992) Probing the interactions of alcohols with biological-membranes with the fluorescent-probe Prodan. Biochemistry 31(39):9473–9481

    Article  PubMed  CAS  Google Scholar 

  32. Karmakar R, Samanta A (2002) Steady-state and time-resolved fluorescence behavior of C153 and PRODAN in room-temperature ionic liquids. J Phys Chem A 106(28):6670–6675

    Article  CAS  Google Scholar 

  33. Marsh D (2009) Reaction fields in the environment of fluorescent probes: polarity profiles in membranes. Biophys J 96(7):2549–2558

    Article  PubMed  CAS  Google Scholar 

  34. Katsaras J, Gutberlet T (2001) Lipid bilayers: Structure and interactions. Springer Verlag, Berlin

    Google Scholar 

  35. Marsh D (1990) CRC handbook of lipids bilayers. Boca Raton

  36. Valeur B (2001) Molecular fluorescence: Principles and applications. Wiley-VCH Verlag GmbH, Weinheim

    Book  Google Scholar 

  37. Jahnig F (1979) Structural order of lipids and proteins in membranes—evaluation of fluorescence anisotropy data. Proc Natl Acad Sci U S A 76(12):6361–6365

    Article  PubMed  CAS  Google Scholar 

  38. Kinosita K, Kawato S, Ikegami A (1977) Theory of fluorescence polarization decay in membranes. Biophys J 20(3):289–305

    Article  PubMed  CAS  Google Scholar 

  39. Rowe BA, Neal SL (2006) Photokinetic analysis of PRODAN and LAURDAN in large unilamellar vesicles from multivariate frequency-domain fluorescence. J Phys Chem B 110(30):15021–15028

    Article  PubMed  CAS  Google Scholar 

  40. Chong PLG, Wong PTT (1993) Interactions of Laurdan with phosphatidylcholine liposomes—a high-pressure ftir study. Biochim Biophys Acta 1149(2):260–266

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by USP, CAPES (Rede nBioNet, 655/09), FAPESP (09/53074-6 and 10/08365-0) and CNPq (MTL research fellowship).We are grateful to Dr. E.L. Duarte for helping with some of the fluorescence experiments.

Author information

Authors and Affiliations

  1. Instituto de Física, Universidade de São Paulo, CP 66318, CEP 05314-970, São Paulo, SP, Brazil

    Cintia C. Vequi-Suplicy & M. Teresa Lamy

  2. Instituto de Física, Universidade Federal de Goiás, CP 131, CEP 74001-970, Goiânia, GO, Brazil

    Cássia A. Marquezin

Authors
  1. Cintia C. Vequi-Suplicy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Teresa Lamy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cássia A. Marquezin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cássia A. Marquezin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vequi-Suplicy, C.C., Lamy, M.T. & Marquezin, C.A. The New Fluorescent Membrane Probe Ahba: A Comparative Study with the Largely Used Laurdan. J Fluoresc 23, 479–486 (2013). https://doi.org/10.1007/s10895-013-1172-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10895-013-1172-3

Keywords

Search

Navigation