Skip to main content
SpringerLink
Log in

pH and temperature dependent relaxation dynamics of Hoechst-33258: a time resolved fluorescence study

  • Paper
  • Published:
Photochemical & Photobiological Sciences Aims and scope Submit manuscript

Abstract

The photophysical behavior of Hoechst 33258 (H33258) in aqueous solution has been studied by steady-state and time-resolved fluorescence measurements. The intriguing intramolecular geometrical orientations of the dye bring out major modulation on its photophysical behavior, especially in the fluorescence emission characteristics with pH. It has been seen that a change in the solution pH from 7 to 4.5 enhances the emission yield by ~20 fold and this change is ~80-fold on changing the pH from 1.5 to 4.5. While a fast flipping motion among the two benzimidazole rings is considered to be one of the most probable mechanisms for the fast fluorescence decay, a more planar structure of the dicationic form at pH 4.5 having a double bond character between the two benzimidazolium groups is suggested to be the most likely fluorescent species. A similar planar structure is in fact considered to be the fluorescent emitting species of H33258 on minor groove binding to DNA. On the basis of temperature dependent fluorescence decay dynamics explored for the dye in solutions at pH 7 and 4.5, it is understood that a nearly isoenergetic double-well excited state potential is possibly involved in the excited state relaxation dynamics of the dye at pH 7. On increasing the temperature, the conversion to the planar structure is facilitated from the non-planar LE state, enhancing the emission probability of the dye.

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

Similar content being viewed by others

References

  1. J. R. Lakowicz, Principles of fluorescence spectroscopy, Springer: New York, 2006.

    Book  Google Scholar 

  2. H. LeVine III, Quantification of beta-sheet amyloid fibril structures with thioflavin T, Methods Enzymol., 1999, 309, 274.

    Article  CAS  Google Scholar 

  3. (a)_H. Naiki, K. Higuchi, M. Hosokawa and T. Takeda, Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavine T, Anal. Biochem., 1989, 177, 244–249

    Article  CAS  Google Scholar 

  4. C. E. Kung and J. K. Reed, Fluorescent molecular rotors: a new class of probes for tubulin structure and assembly, Biochemistry, 1989, 28, 6678–6686.

    Article  CAS  Google Scholar 

  5. G. Cosa, K.-S. Focsaneanu, J. R. N. McLean, J. P. McNamee and J. C. Scaiano, Photophysical properties of fluorescenct DNA-dyes bound to single and double-stranded DNA in aqueous buffered solution, Photochem. Photobiol., 2001, 73, 585–599.

    Article  CAS  Google Scholar 

  6. A. C. Bhasikuttan, J. Mohanty, W. M. Nau and H. Pal, Efficient fluorescence enhancement and cooperative binding of an organic dye in a supra-biomolecular host-protein assembly, Angew. Chem., Int. Ed., 2007, 46, 4120–4122.

    Article  CAS  Google Scholar 

  7. A. C. Bhasikuttan, J. Mohanty and H. Pal, Interaction of Malachite Green with Guanine-Rich Single Strand DNA: Preferential Binding to G-Quadruplex, Angew. Chem., Int. Ed., 2007, 46, 9305–9307.

    Article  CAS  Google Scholar 

  8. S. Dutta Choudhury, J. Mohanty, H. Pal and A. C. Bhasikuttan, Cooperative Metal Ion Binding to a Cucurbit[7]uril-Thioflavin T Complex: Demonstration of a Stimulus Responsive Fluorescent Supramolecular Capsule, J. Am. Chem. Soc., 2010, 132, 1395–1401.

    Article  Google Scholar 

  9. F. G. Loontiens, P. Regenfuss, A. Zechel, L. Dumortier and R. M. Clegg, Binding characteristics of Hoechst 33258 with calf thymus DNA, Poly[d(A-T)], and d(CCGGAATTCCGG): Multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities, Biochemistry, 1990, 29, 9029–9039.

    Article  CAS  Google Scholar 

  10. J.-H. Moon, S. K. Kim, U. Sehlstedt, A. Rodger and B. Norden, DNA structural features responsible for sequence dependent binding geometries of Hoechst 33258, Biopolymers, 1996, 38, 593–606.

    Article  CAS  Google Scholar 

  11. L. Goracci, R. Germani, G. Savelli and D. M. Bassani, Hoechst 33258 as a pH-sensitive probe to study the interaction of amine oxide surfactants with DNA, ChemBioChem., 2005, 6, 197–203.

    Article  CAS  Google Scholar 

  12. H. Ojha, B. M. Murari, S. Anand, M. I. Hassan, F. Ahmad and N. K. Chaudhury, Interaction of DNA minor groove binder Hoechst 33258 with Bovine Serum Albumin, Chem. Pharm. Bull., 2009, 57, 481–486.

    Article  CAS  Google Scholar 

  13. N. V. Lyubimova, P. G. Coultas, K. Yuen and R. F. Martin, In vivo radioprotection of mouse brain endothelial cells by Hoechst 33342, Br. J. Radiol., 2001, 74, 77–82.

    Article  CAS  Google Scholar 

  14. L. Denison, A. Haigh, G. D’Cunha and R. F. Martin, DNA Ligands as Radioprotectors: Molecular Studies with Hoechst 33342 and Hoechst 33258, Int. J. Radiat. Biol., 1992, 61, 69–74.

    Article  CAS  Google Scholar 

  15. T. R. Downs and W. W. Wilfinger, Fluorometric quantification of DNA in cells and tissues, Anal. Biochem., 1983, 131, 538–547.

    Article  CAS  Google Scholar 

  16. C. F. Cesarone, C. Bolognesi and L. Santi, Improved microfluorometric DNA determination in biological material using 33258 Hoechst, Anal. Biochem., 1979, 100, 188–197.

    Article  CAS  Google Scholar 

  17. D. L. Stout and F. F. Becker, Fluorometric quantitation of single-stranded DNA: A method applicable to the technique of alkaline elution, Anal. Biochem., 1982, 127, 302–307.

    Article  CAS  Google Scholar 

  18. M. Ladinig, W. Leupin, M. Meuwly, M. Respondek, J. Wirz and V. Zoete, Protonation equilibria of Hoechst-33258 in aqueous solution, Helv. Chim. Acta, 2005, 88, 53–67.

    Article  CAS  Google Scholar 

  19. K. Kalninsh, D. V. Pestov and Y. K. Roshchina, Absorption and fluorescence spectra of the probe Hoechst 33258, J. Photochem. Photobiol., A, 1994, 83, 39–47.

    Article  CAS  Google Scholar 

  20. T. Stokke and H. B. Steen, Multiple binding modes for Hoechst 33258 to DNA, J. Histochem. Cytochem., 1985, 33, 333–338.

    Article  CAS  Google Scholar 

  21. H. Gorner, Direct and sensitized photoprocesses of bis-benzimidazole dyes and the effects of surfactants and DNA, Photochem. Photobiol., 2001, 73, 339–348.

    Article  CAS  Google Scholar 

  22. M. Rahimian, Y. Miao and W. D. Wilson, Influence of DNA structure on adjacent site cooperative binding, J. Phys. Chem. B, 2008, 112, 8770–8778.

    Article  CAS  Google Scholar 

  23. Y. Guan, R. Shi, X. Li, M. Zhao and Y. Li, Multiple binding modes for dicationic Hoechst 33258 to DNA, J. Phys. Chem. B, 2007, 111, 7336–7344.

    Article  CAS  Google Scholar 

  24. A. Adhikary, V. Buschmann, C. Muller and M. Sauer, Ensemble and single-molecule fluorescence spectroscopic study of binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA, Nucleic Acids Res., 2003, 31, 2178–2186.

    Article  CAS  Google Scholar 

  25. S. K. Pal, L. Zhao and A. H. Zewail, Water at DNA surfaces: Ultrafast dynamics in minor groove recognition, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 8113–8118.

    Article  CAS  Google Scholar 

  26. D. Banerjee and S. K. Pal, Ultrafast charge transfer and solvation of DNA minor groove binder: Hoechst 33258 in restricted environments, Chem. Phys. Lett., 2006, 432, 257–262.

    Article  CAS  Google Scholar 

  27. V. N. Umetskaya and Y. M. Rozanov, Mechanism of the interaction of DNA with the fluorescent dye Hoechst 33258, Biophysika, 1990, 35, 399–401.

    CAS  Google Scholar 

  28. K. E. Furse and S. A. Corcelli, The dynamics of water at DNA interfaces: computational studies of Hoechst 33258 bound to DNA, J. Am. Chem. Soc., 2008, 130, 13103–13109.

    Article  CAS  Google Scholar 

  29. M. Shaikh, J. Mohanty, A. C. Bhasikuttan, V. D. Uzunova, W. M. Nau and H. Pal, Salt-induced Guest Relocation from a Supramolecular Cavity into a Biomolecular Pocket: Interplay between Cucurbit[7]uril and Albumin, Chem. Commun., 2008, 3681–3683.

    Google Scholar 

  30. M. J Frisch, G. W. Trucks, M. Head-Gorden, P. M. W. Gill, M. W. Wong, J. B. Foresman, B. G. Johnson, H. B. Schlegel, M. A. Robb, E. S. Replogle, R. Gomperts, J. L. Andres, K. Rahavachari, J. S. Binkley, C. Gonzalez, R. Martin, L. D. J. Fox, D. J. Defrees, J. Baker, J. J. P. Stewart, J. A. Pople, Gaussian 92, Gaussian, Inc, Pittsburgh, PA, 1992.

    Google Scholar 

  31. J. F. G. Walz, B. Terenna and D. Rolinee, Equilibrium studies on neutral red-DNA binding, Biopolymers, 1975, 14, 825–837.

    Article  CAS  Google Scholar 

  32. J. Mohanty, A. C. Bhasikuttan, W. M. Nau and H. Pal, Host–guest complexation of neutral red with macrocyclic host molecules: Contrasting pKa shifts and binding affinities for cucurbit[7] uril and b-cyclodextrin, J. Phys. Chem. B, 2006, 110, 5132–5138.

    Article  CAS  Google Scholar 

  33. M. Shaikh, J. Mohanty, P. K. Singh, W. M. Nau and H. Pal, Complexation of Acridine Orange by Cucurbit[7]uril and beta-cyclodextrin: Photophysical Effects and pKa Shifts, Photochem. Photobiol. Sci., 2008, 7, 408–414.

    Article  CAS  Google Scholar 

  34. R. L. Jones and W. D. Wilson, Effect of ionic strength on the pKa of ligands bound to DNA, Biopolymers, 1981, 20, 141–154.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India

    Nilotpal Barooah, Jyotirmayee Mohanty, Haridas Pal, Sisir K. Sarkar, Tulsi Mukherjee & Achikanath C. Bhasikuttan

Authors
  1. Nilotpal Barooah
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jyotirmayee Mohanty
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Haridas Pal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sisir K. Sarkar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tulsi Mukherjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Achikanath C. Bhasikuttan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Achikanath C. Bhasikuttan.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Barooah, N., Mohanty, J., Pal, H. et al. pH and temperature dependent relaxation dynamics of Hoechst-33258: a time resolved fluorescence study. Photochem Photobiol Sci 10, 35–41 (2011). https://doi.org/10.1039/c0pp00215a

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1039/c0pp00215a

Search

Navigation