Journal of Fluorescence

, Volume 18, Issue 2, pp 519–526 | Cite as

A Fluorescence Analysis of ANS Bound to Bovine Serum Albumin: Binding Properties Revisited by Using Energy Transfer

Original Paper

Abstract

Determination of binding parameters such as the number of ligands and the respective binding constants require a considerable number of experiments to be performed. These involve accurate determination of either free and/or bound ligand concentration irrespective of the measurement technique applied. Then, an appropriate theoretical model is used to fit the experimental data, and to extract the binding parameters. In this work, the interaction between bovine serum albumin (BSA) and 1-anilino-8-naphthalene sulphonate (ANS) is revisited. Using steady state fluorescence spectroscopy, the binding isotherm of BSA/ANS was obtained applying the Halfman–Nishida approach. The binding parameters, site number, and binding site association constants, were determined from the stoichiometric Adair model and Job’s plot. The binding parameters obtained were then correlated to the distance of the respective binding site to the tryptophan residues using the energy transfer technique. This approach, that uses both tryptophans independently from each other, is presented as a tool to help understand the binding mechanism of the albumin fluorescent complex. The results show that ANS molecules bind to BSA in up to five different binding sites. Energy transfer from the tryptophan residues to the BSA/ANS complex shows that the four highest affinity binding sites (>104 M−1) are located at a reasonably close distance (18–27 Å) to at least one of two tryptophan residues, while the lowest affinity binding site (~104 M−1) is located over 34 Å away from the both tryptophans.

Keywords

Bovine serum albumin 1-anilino-8-naphthalene sulfonate Ligand binding Energy transfer 

Notes

Acknowledgement

This work was supported by Science Foundation Ireland under Grant number (02/IN.1M231)

References

  1. 1.
    He XM, Carter DC (1992) Atomic-structure and chemistry of human serum-albumin. Nature 358:209–215PubMedCrossRefGoogle Scholar
  2. 2.
    Peters T (1985) Serum albumin. Adv Protein Chem 37:161–245PubMedGoogle Scholar
  3. 3.
    Kragh-Hansen U, Chuang VTG, Otagiri M (2002) Practical aspects of the ligand-binding and enzymatic properties of human serum albumin. Bio Pharm Bull 25:695–704CrossRefGoogle Scholar
  4. 4.
    Harding SE, Chowdhry BZ (2001) Protein-ligand interactions: structure and spectroscopy. Oxford University Press, New YorkGoogle Scholar
  5. 5.
    Pacifici GM, Viani A (1992) Methods of determining plasma and tissue binding of drugs—pharmacokinetic consequences. Clin Pharmacokinet 23:449–468PubMedGoogle Scholar
  6. 6.
    Georgiou ME, Georgiou CA, Koupparis MA (1999) Automated flow injection gradient technique for binding studies of micromolecules to proteins using potentiometric sensors: application to bovine serum albumin with anilinonaphthalenesulfonate probe and drugs. Anal Chem 71:2541–2550PubMedCrossRefGoogle Scholar
  7. 7.
    Birdsall B, King RW, Wheeler MR, Lewis CA, Goode SR, Dunlap RB, Roberts GCK (1983) Correction for light-absorption in fluorescence studies of protein-ligand interactions. Anal Biochem 132:353–361PubMedCrossRefGoogle Scholar
  8. 8.
    Eftink MR (1997) Fluorescence methods for studying equilibrium macromolecule-ligand interactions. Meth Enz 278:221–257CrossRefGoogle Scholar
  9. 9.
    Weber G, Young LB (1964) Fragmentation of bovine serum albumin by pepsin.1. Origin of acid expansion of albumin molecule. J Biol Chem 239:1415–1423PubMedGoogle Scholar
  10. 10.
    Cardamone M, Puri NK (1992) Spectrofluorometric assessment of the surface hydrophobicity of proteins. Biochem J 282:589–593PubMedGoogle Scholar
  11. 11.
    Haskard CA, Li-Chan ECY (1998) Hydrophobicity of bovine serum albumin and ovalbumin determined using uncharged (PRODAN) and anionic (ANS(-)) fluorescent probes. J Agric Food Chem 46:2671–2677CrossRefGoogle Scholar
  12. 12.
    Hazra P, Chakrabarty D, Chakraborty A, Sarkar N (2004) Probing protein-surfactant interaction by steady state and time-resolved fluorescence spectroscopy. Biochem Biophys Res Commun 314:543–549PubMedCrossRefGoogle Scholar
  13. 13.
    Bagatolli LA, Kivatinitz SC, Fidelio GD (1996) Interaction of small ligands with human serum albumin IIIA subdomain. How to determine the affinity constant using an easy steady state fluorescent method. J Pharm Sci 85:1131–1132PubMedCrossRefGoogle Scholar
  14. 14.
    Matulis D, Lovrien R (1998) 1-anilino-8-naphthalene sulfonate anion-protein binding depends primarily on ion pair formation. Biophys J 74:422–429PubMedGoogle Scholar
  15. 15.
    Naik DV, Paul WL, Threatte RM, Schulman SG (1975) Fluorometric-determination of drug-protein association constants—binding of 8-anilino-1-naphthalenesulfonate by bovine serum-albumin. Anal Chem 47:267–270CrossRefGoogle Scholar
  16. 16.
    Huang CY (1982) Determination of binding stoichiometry by the continuous variation method: the job plot. Meth Enz 87:509–525Google Scholar
  17. 17.
  18. 18.
    van Holde KE, Johnson WC, Ho PS (1998) Principles of physical biochemistry. Prentice Hall, Englewood Cliffs, NJGoogle Scholar
  19. 19.
    Scatchard G (1949) The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660–672CrossRefGoogle Scholar
  20. 20.
    Fletcher JE, Spector AA, Ashbrook JD (1970) Analysis of macromolecule-ligand binding by determination of stepwise equilibrium constants. Biochem 9:4580–4587CrossRefGoogle Scholar
  21. 21.
    Klotz IM (1974) Protein interactions with small molecules. Acc Chem Res 7:162–168CrossRefGoogle Scholar
  22. 22.
    Klotz IM, Hunston DL (1984) Mathematical models for ligand-receptor binding. Real sites, ghost sites. J Biol Chem 259:60–62Google Scholar
  23. 23.
    Halfman CJ, Nishida T (1972) Method for measuring the binding of small molecules to proteins from binding-induced alterations of physical–chemical properties. Biochem 11:3493–3498CrossRefGoogle Scholar
  24. 24.
    Lipskier JF, Tran-Thi TH (1993) Supramolecular assemblies of porphyrins and phthalocyanines bearing oppositely charged substituents—1st evidence of heterotrimer formation. Inorg Chem 32:722–731CrossRefGoogle Scholar
  25. 25.
    De S, Girigoswami A, Das S (2004) Fluorescence probing of albumin–surfactant interaction. J Coll Int Sci 285:562–573CrossRefGoogle Scholar
  26. 26.
    Brocklehurst JR, Freedman RB, Hancock DJ, Radda GK (1970) Membrane studies with polarity-dependant and excimer-forming fluorescent probes. Biochem J 116:721–731PubMedGoogle Scholar
  27. 27.
    Axelsson I (1978) Characterization of proteins and other macromolecules by agarose-gel chromatography. J Chromatog 152:21–32CrossRefGoogle Scholar
  28. 28.
    Putnam FW (1975) The plasma proteins: structure, function and genetic control, vol 1, 2nd edn. Academic, New YorkGoogle Scholar
  29. 29.
    Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Academic, New YorkGoogle Scholar
  30. 30.
    Moens PDJ, Helms MK, Jameson DM (2004) Detection of tryptophan to tryptophan energy transfer in proteins. Protein Journal 23:79–83PubMedCrossRefGoogle Scholar
  31. 31.
    Petitpas I, Grune T, Bhattacharya AA, Curry S (2001) Crystal structures of human serum albumin complexed with monounsaturated and polyunsaturated fatty acids. J Mol Biol 314:955–960 (PDB ID: 1gnj)PubMedCrossRefGoogle Scholar
  32. 32.
    Ghuman J, Zunszain PA, Petitpas I, Bhattacharya AA, Otagiri M, Curry S (2005) Structural basis of the drug-binding specificity of human serum albumin. J Mol Bio 353:38–52CrossRefGoogle Scholar
  33. 33.
    Bagatolli LA, Kivatinitz SC, Aguilar F, Soto MA, Sotomayor P, Fidelio GD (1996) Two distinguishable fluorescent modes of 1-anilino-8-naphtalenesulfonate bound to human albumin. J Fluoresc 6:33–40CrossRefGoogle Scholar
  34. 34.
    Togashi DM, Ryder AG (2006) Time-resolved fluorescence studies on bovine serum albumin denaturation process. J Fluoresc 16:153–160PubMedCrossRefGoogle Scholar
  35. 35.
    Teale FWJ (1960) The ultraviolet fluorescence of proteins in neutral solution. Biochem J 76:381–388PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.Nanoscale Biophotonics Laboratory, Department of ChemistryNational University of Ireland, GalwayGalwayIreland
  2. 2.National Centre for Biomedical Engineering ScienceNational University of Ireland, GalwayGalwayIreland

Personalised recommendations