Journal of Fluorescence

, Volume 20, Issue 5, pp 993–1002 | Cite as

Photophysical Studies on the Interaction of Acridinedione Dyes with Universal Protein Denaturant: Guanidine Hydrochloride

  • R. Kumaran
  • T. Varalakshmi
  • E. J. Padma Malar
  • P. Ramamurthy
Original Paper


Photophysical studies of photoinduced electron transfer (PET) and non-PET based acridinedione dyes with guanidine hydrochloride (GuHCl) were carried out in water and methanol. Addition of GuHCl to photoinduced electron transfer (PET) based acridinedione dye (ADR 1) results in a fluorescence enhancement, whereas a non-PET based dye (ADR 2) shows no significant change in the fluorescence intensity and lifetime. Addition of GuHCl to ADR 1 dye in methanol results in single exponential decay behaviour, on the contrary a biexponential decay pattern was observed on the addition of GuHCl in water. Absorption and emission spectral studies of ADR 1 dye interaction with GuHCl reveals that the dye molecule is not in the protonated form in aqueous GuHCl solution, and the dye is confined to two distinguishable microenvironment in the aqueous phase. A large variation in the microenvironment around the dye molecule is created on the addition of GuHCl and this was ascertained by time-resolved area normalized emission spectroscopy (TRANES) and time-resolved emission spectroscopy (TRES). The dye molecule prefers to reside in the hydrophobic microenvironment, rather in the hydrophilic aqueous phase is well emphasized by time-resolved fluorescence lifetime studies. The mechanism of fluorescence enhancement of ADR 1 dye by GuHCl is attributed to the suppression of the PET process occuring through space.


Acridinedione dyes Guanidine hydrochloride Photoinduced electron transfer Fluorescence enhancement Hydrogen-bonding 



Financial support by DST-IRHPA and UGC-INNOVATIVE Programme is acknowledged. R.K thanks the UGC for providing the financial assistance.

Supplementary material

10895_2010_646_MOESM1_ESM.doc (43 kb)
Table S1 (DOC 43 kb)


  1. 1.
    Creighton TE (1992) Protein folding. Freeman, New YorkGoogle Scholar
  2. 2.
    Joly M (1965) A physiochemical approach on the denaturation of proteins. Academic, LondonGoogle Scholar
  3. 3.
    Michnik A, Sulkowska A (1997) Hydrogen-bonded interactions in alkylurea-and amide-D2O-gunanidine.HCl systems. J Mol Struct 410–411:17–21Google Scholar
  4. 4.
    Sato S, Sayid CJ, Raleigh DP (2003) The failure of simple empirical relationships to predict the viscocity of mixed aqueous solution of guanidine hydrochloride and glucose has important implications for the study of protein folding in [In Process Citation]. Protein Sci 9:1601–1603CrossRefGoogle Scholar
  5. 5.
    Kawahara K, Tanford C (1966) Viscocity and density of aqueous solution of urea and guanidine hydrochloride. J Biol Chem 241:3228–3232PubMedGoogle Scholar
  6. 6.
    Moosavi-Movahedi AA, Naderi GA, Farzami B (1994) The denaturation behaviour of calmodulin in sodium n-dodecyl sulphate, dodecyl trimethyl ammonium bromide, guanidine hydrochloride and urea. Thermochim Acta 239:61–71CrossRefGoogle Scholar
  7. 7.
    Morjana NA, McKeone BJ, Gilbert HF (1993) Guanidine hydrochloride stabilization of a partially unfolded intermediate during the reversible denaturation of protein disulfide isomerase. Proc Natl Acad Sci USA 90:2107–2111CrossRefPubMedGoogle Scholar
  8. 8.
    Mukhopadhyay A (1997) Inclusion bodies and purification of proteins in biologically active forms. Adv Biochem Eng Biotechnol 56:61–109PubMedGoogle Scholar
  9. 9.
    Rudolph R, Lilie H (1996) In vitro folding of inclusion body proteins. FASEB J 10:49–56PubMedGoogle Scholar
  10. 10.
    Levine AD, Rangwala SH, Peel MA, Leimgruber RM, Manning JA, Bishop BF, Olins PO (1995) High level expression and refolding of mouse interleukin 4 synthesized in escherichia coli. J Biol Chem 270:7445–7452CrossRefPubMedGoogle Scholar
  11. 11.
    Cox RA (1968) The use of guanidinium chloride in the isolation of nucleic acids. In: Grossman L, Moldave K (eds) Methods in enzymology, vol 12. Academic, New York, pp 120–129Google Scholar
  12. 12.
    Sarkar N, Bhattacharyya K (1991) Effect of urea on micelles: fluorescence of P-toluidinonaphthalenesulphonate. Chem Phys Lett 180:283–286CrossRefGoogle Scholar
  13. 13.
    Briganti G, Puvvada S, Blankschtein D (1991) Effect of urea on the micellar properties of aqueous solutions of non-ionic surfactants. J Phys Chem 95:8989–8995CrossRefGoogle Scholar
  14. 14.
    Baglioni P, Rivara-Minten E, Dei L, Ferroni E (1990) ESR study of sodium dodecyl sulfate and dodecyltrimethylammonium bromide micellar solutions: effect of urea. J Phys Chem 94:8218–8222CrossRefGoogle Scholar
  15. 15.
    Garcia-Rio L, Leis JR, Mejuto JC, Elena Pena M, Iglesias E (1994) Effects of additives on the internal dynamics and properties of water/AOT/isooctane microemulsions. Langumir 10:1676–1683CrossRefGoogle Scholar
  16. 16.
    Godinez LA, Patel S, Criss CM, Kaifer AE (1995) Calorimetric studies on the complexation of several ferrocene derivatives by.alpha.- and.beta.- cyclodextrin. Effects of urea on the thermodynamic parameters. J Phys Chem 99:17449–17455CrossRefGoogle Scholar
  17. 17.
    Sarkar N, Das K, Nath D, Bhattacharyya K (1992) Interaction of urea with fluorophores bound to cyclodextrins. Fluorescence of p-toluidino naphthalene sulphonate interaction of urea with fluorophores bound to cyclodextrins. Chem Phys Lett 181:491–496CrossRefGoogle Scholar
  18. 18.
    Shen X, Belletête M, Durocher G (1997) Studies of the inclusion complexation between a 3H-indole and β-cyclodextrin in the presence of urea, Sodium Dodecyl Sulfate, and 1-Propanol. Langumir 13:5830–5836CrossRefGoogle Scholar
  19. 19.
    Shen X, Belletête M, Durocher G (1997) Quantitative study of the hydrophobic interaction mechanism between urea and molecular probes used in sensing some microheterogeneous media. J Phys Chem B 101:8212–8220CrossRefGoogle Scholar
  20. 20.
    Castellino FJ, Barker R (1968) The denaturing effectiveness of guanidinium, carbamoylguanidinium, and guanylguanidinium salts. Biochemistry 7:4135–4138CrossRefPubMedGoogle Scholar
  21. 21.
    Kalyanasundaram K (1987) Photochemistry in microheterogeneous system. Academic, New YorkGoogle Scholar
  22. 22.
    Lakowicz JR (1999) Principles of fluorescence spectroscopy, 3rd edn. Kluwer Academic/Plenum, New YorkGoogle Scholar
  23. 23.
    Singh S, Chhina S, Sharma VK, Sachev SS (1982) Cationic hydrogenation of benzyl alcohols and arylethylenes using acridane derivatives as hindered NADH model. J Chem Soc Chem Commun 8:453–459CrossRefGoogle Scholar
  24. 24.
    Selvaraju C, Ramamurthy P (2004) Excited-state behavior and photoionization of 1, 8-acridinedione dyes in micelles–comparison with NADH oxidation. Chem Eur J 10:2253–2262CrossRefGoogle Scholar
  25. 25.
    Stryer L (1995) Biochemistry, 4th edn. Freeman, New YorkGoogle Scholar
  26. 26.
    Thiagarajan V, Selvaraju C, Ramamurthy P (2003) Excited state behaviour of acridinedione dyes in PMMA matrix: inhomogeneous broadening and enhancement of triplet. J Photochem Photobiol A Chem 157:23–31CrossRefGoogle Scholar
  27. 27.
    Indirapriydharshini VK, Karunanithi P, Ramamurthy P (2001) Inclusion of resorcinol-based acridinedione dyes in cyclodextrins: Fluorescence enhancement. Langmuir 17:4056–4060CrossRefGoogle Scholar
  28. 28.
    Thiagarajan V, Indirapriyadharsini VK, Ramamurthy P (2006) Fencing of photoinduced electron transfer in nonconjugated bichromophoric system by β-cyclodextrin nanocavity. J Incl Phenom and Macrocyclic chemistry 56:309–313CrossRefGoogle Scholar
  29. 29.
    Kumaran R, Ramamurthy P (2006) PET suppression of acridinediones by urea derivatives in water and methanol. J Phys Chem B 110:23783–23789CrossRefPubMedGoogle Scholar
  30. 30.
    Kumaran R, Ramamurthy P Photophysical studies of PET based acridinedionedyes with globular protein: Bovine Serum Albumin (BSA), Accepted for Publication in J. Luminescence (Lumin0-08-00192)Google Scholar
  31. 31.
    Srividya N, Ramamurthy P, Ramakrishnan VT (1997) Solvent effects on the absorption and fluorescence spectra of some acridinedione dyes: determination of ground and excited state dipole moments. Spectrochim Acta Part A 53:1743–1753CrossRefGoogle Scholar
  32. 32.
    Srividya N, Ramamurthy P, Ramakrishnan VT (1998) Photophysical studies of acridine (1, 8) dione dyes; a new class of laser dyes. Spectrochim Acta Part A 54:245–253CrossRefGoogle Scholar
  33. 33.
    Srividya N, Ramamurthy P, Shanmugasundaram P, Ramakrishnan VT (1996) Synthesis, characterisation and electrochemistry of some acridine-1, 8- dione dyes. J Org Chem 61:5083–5089CrossRefGoogle Scholar
  34. 34.
    Koti ASR, Krishna MMG, Periasamy N (2001) Time resolved area normalized emission spectroscopy (TRANES): A novel method for confirming emission from two excited states. J Phys Chem A 105:1767–1771CrossRefGoogle Scholar
  35. 35.
    Koti ASR, Periasamy N (2001) Application of time resolved area normalized emission spectroscopy to multi component systems. J Chem Phys 115:7094–7099CrossRefGoogle Scholar
  36. 36.
    Connor DV, Phelps D (1984) Time correlated single photon counting. Academic, LondonGoogle Scholar
  37. 37.
    Periasamy N, Doraiswamy S, Maiya GB, Venkataraman B (1988) Fluorescence quenching of cationic dyes by charged quenching. J Chem Phys 88:1638–1642CrossRefGoogle Scholar
  38. 38.
    Bankar KV, Bhagat VR, Das R, Doraiswamy S, Ghangrekar AS, Kamat DS, Periasamy N, Srivatsavoy VJP, Venkataraman B (1989) Techniques for the study of fast chemical processes with half-times of the order of microseconds or less Indian. J Appl Phys 27(7–8):416–428Google Scholar
  39. 39.
    Laws WR, Brand L (1979) Analysis of two-state excited-state. The fluorescence decay of 2-Naphthol. J Phys Chem 83:795–802CrossRefGoogle Scholar
  40. 40.
    Venkatachalapathy B, Ramamurthy P (1999) Excited state proton and electron transfer reactions of acridinedione dyes with amines. Phys Chem Chem Phys 1:2223–2230CrossRefGoogle Scholar
  41. 41.
    Srividhya N, Ramamurthy P, Ramakrishnan VT (2000) Photooxidation of acridine(1, 8)dione dyes: flash photolysis investigation of the mechanistic details. Phys Chem Chem Phys 2:5120–5126CrossRefGoogle Scholar
  42. 42.
    Katz S (1968) Partial molar volume and conformational changes produced by the denaturation of albumin by guanidine hydrochloride. Biochim Biophys Acta 154(3):468–477PubMedGoogle Scholar
  43. 43.
    Makhatadze GI, Privalov PL (1992) Protein interactions with urea and guanidinium chloride: a calorimetric study. J Mol Biol 226:491–505CrossRefPubMedGoogle Scholar
  44. 44.
    Hedwig GR, Lilley TH, Linsdell H (1991) Calorimetric and volumetric studies of the interactions of some amides in water and in 6 mol dm−3 aqueous guanidinium chloride. J Chem Soc Faraday Trans 87:2975–2982CrossRefGoogle Scholar
  45. 45.
    Lee C, Yang W, Parr RG (1988) Development of the colle-salvetti correlation-energy formula into a functional of the electron density. Phys Rev B 37:785–789CrossRefGoogle Scholar
  46. 46.
    Gaussian 03 (2004) Revision B.04. Gaussian Inc, Wallingford CTGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • R. Kumaran
    • 1
  • T. Varalakshmi
    • 1
  • E. J. Padma Malar
    • 1
  • P. Ramamurthy
    • 1
  1. 1.National Centre for Ultrafast ProcessesUniversity of MadrasChennaiIndia

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