European Biophysics Journal

, Volume 14, Issue 1, pp 29–35 | Cite as

Fluorescence of 3-keto-steroids in aqueous solution

Probes for steroid-protein interactions
  • M. Kempfle
  • R. Müller
  • R. Palluk
  • K. A. Zachariasse


The physiologically important 3-keto-steroids are non-fluorescent or only weakly fluorescent in protic as well as in aprotic solvents. In contrast, the 4,6,8(14)-triene-3-one steroids are highly fluorescent in aqueous solution but they do not appreciably fluoresce in other solvents. Evidence is presented that the introduction of double bonds into the skeleton of the 3-keto-steroids leads to a decrease of the energy of the lowest π − π* state, bringing this level into the neighbourhood of the non-fluorescent n − π* state. As a consequence, for two states of approximately the same energy, relatively small perturbations such as those due to solvent interactions, protein binding and micelle formation, will then determine whether a system will fluoresce (π − π* state lowest) or not (n − π* state lowest). When the fluorescent 3-keto-steroids, having three conjugated double bonds, bind to proteins, the fluorescence intensity becomes almost zero, making these compounds useful as probes for steroid-protein interactions. This quenching of the fluorescence is explained by a decrease in energy of the n − π* state relative to the π − π* state of the steroids due to hydrophobic interactions with the proteins.

Key words

Fluorescent steroid probes steroid-protein interactions, energy alternation of n − π* and π − π* states (level crossing) 



6,8-bisdehydrotestosterone; DMSO, dimethylsulfoxide


high pressure liquid chromatography


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Becker RS, Kasha M (1955) Luminescence spectroscopy of porphyrin-like molecules including the chlorophylls. J Am Chem Soc 77:3669–3670Google Scholar
  2. Birks JB (1977) In: Mielenz KD, Velapoldi RA, Mavrodineanu R (eds) Standardisation in spectrometry and luminescence measurements National Bureau of Standards, WashingtonGoogle Scholar
  3. Bredereck K, Förster Th, Oesterlin HG (1962) In: Kallmann HP, Spruch GM (eds) Luminescence of organic and inorganic materials. Wiley, New York, pp 161–175Google Scholar
  4. Chen M, Grätzel M, Thomas JK (1975) Kinetic studies in bile acid micelles. J Am Chem Soc 97:2052–2057Google Scholar
  5. Dederen JC, Coosemans L, De Schryver FC, Van Dormael A (1979) Complex solvent dependence of pyrenealdehyde fluorescence as a micellar polarity probe. Photochem Photobiol 30:443–447Google Scholar
  6. Demas JN, Crosby GA (1971) The measurement of photoluminescence quantum yields. A review. J Phys Chem 75: 991–1024Google Scholar
  7. Inuzuka S, Becker RS (1974) Theoretical consideration of lower excited states of trans-polyene carbaldehydes. I. Electronic structure and oscillator strength of π − π * transition. Bull Chem Jpn 47:88–91Google Scholar
  8. Inuzuka S (1975) Theoretical consideration of lower excited states of trans-polyene carbaldehydes. II. Radiative and non-radiative properties. Bull Chem Soc Jpn 48:779–782Google Scholar
  9. Kasha M (1949) Characterization of electronic transitions in complex molecules. Disc Faraday Soc 9:14–19Google Scholar
  10. Kempfle M, Müller R, Palluk R (1984) Fluoreszierende Steroide-Wechselwirkungen mit Cyclodextrinen Fresenius Z Anal Chem 317:700–701Google Scholar
  11. Kempfle M, Breuer H, Müller R, Palluk R (1985) Fluorescent steroids and their interactions with 3α-and β-hydroxysteroid dehydrogenases. Acta Endocrinol Suppl 108: 112–113Google Scholar
  12. Mataga N, Kubota K (1970) Molecular interactions and electronic spectra. Marcel Dekker, New York, p 355Google Scholar
  13. Müller R, Palluk R, Kempfle M (1982) Charakterisierung der Fluoreszenzeigenschaften von 4,6,8(14)-Trien-3-on-Steroiden. Fresenius Z Anal Chem 311:369–370Google Scholar
  14. Müller R, Palluk R, Kempfle M (1983) 4,6,8(14)-trien-3-one steroids as fluorescing components in preparations of 4,6-diene-3-one steroids. Arch Pharm, 316:91–92Google Scholar
  15. Müller R, Palluk R, Ehlenz K, Kempfle M (1985) Fluorescent steroids: Homogeneous fluorescence immunoassay of cortisol in plasma by means of 17α-hydroxy-4,6,8(14) pregnatriene-3,20-dione. Acta Endocrinol Suppl 108:115–116Google Scholar
  16. Müller R, Palluk R, Kempfle M (1986) Fluoreszierende 4,6,8(14)-Trien-3-on Steroide als Indikatoren zur Untersuchung der Proteinbindung von Steroiden. Stud Biophys 109 (in press)Google Scholar
  17. Palluk R (1983) Protein-Wechselwirkungen fluoreszierender Steroide. Thesis, University of BonnGoogle Scholar
  18. Palluk R, Müller R, Kempfle M (1982) Untersuchung von Steroid-Protein-Wechselwirkungen mit Hilfe der Fluoreszenz von 4,6,8(14)-Trien-3-on-Steroiden. Fresenius Z Anal Chem 311:370–371Google Scholar
  19. Schröder F (1984) Fluorescent sterols: Probe molecules of membrane structure and function. Prog Lipid Res 23: 97–113Google Scholar
  20. Schulte KE, Rücker G, Fachmann H (1968) Ergosta 4,6,8(14), 22-Tetraenon-(3) als Inhaltstoff des Lärchenschwammes. Tetrahedron Lett 46:4763–4764Google Scholar
  21. Westphal U (1978) Bindung von Progesteron und anderen Steroidhormonen and Proteine des Blutserums. Hoppe-Seyler's Z Physiol Chem 359: 431–447Google Scholar
  22. White JD, Taylor SI (1970) Biosynthesis of ergosta-4,6,8(14), 22-tetraene-3-one. In vivo incorporation of a 1,4-dioxide. J Am Chem Soc 92:5811–5813Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • M. Kempfle
    • 1
  • R. Müller
    • 1
  • R. Palluk
    • 1
  • K. A. Zachariasse
    • 2
  1. 1.Physiologisch-chemisches Institut der Universität BonnBonnFederal Republic of Germany
  2. 2.Abteilung SpektroskopieMax-Planck-Institut für biophysikalische ChemieGöttingenFederal Republic of Germany

Personalised recommendations