Determining the Fluorescence Spectrum of a Protein

Roger H. Pain1

1 Jozef Stefan Institute, Ljubljana
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.7
DOI:  10.1002/0471140864.ps0707s38
Online Posting Date:  January, 2005
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Fluorescence spectra of proteins are determined chiefly by the polarity of the environment of the tryptophan and tyrosine residues and by their specific interactions. A thorough consideration of fluorescence spectrometers and their calibration is provided along with important information regarding spectrometer cells, buffers and clarification of samples. Protocols are provided for recording fluorescence spectra and for measuring fluorescence quenching to probe the accessibility of tryptophan residues to small molecules (to yield information about the structural environment of the tryptophan). The technique involves quantifying the decrease in protein fluorescence intensity in the presence of increasing concentrations of quencher, followed by analysis of the data to give details of the interaction of the quencher with the tryptophan residue. Finally, a gives details on how to interpret fluorescence spectra.

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Recording a Fluorescence Emission Spectrum
  • Basic Protocol 2: Determination of Fluorescence Quenching
  • Support Protocol 1: Basic Theory and Interpretation of Fluorescence Spectra
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Recording a Fluorescence Emission Spectrum

  • Calibrated fluorescence spectrometer, buffer solution, and cleaned cuvettes (see Strategic Planning)
  • Clarified protein solution of known concentration (see Strategic Planning for clarification methods and unit 7.2 for concentration determination) with absorbance usually in the range of 0.05 to 0.1 at the wavelength to be used for excitation

Basic Protocol 2: Determination of Fluorescence Quenching

  • Protein solution in buffer, A 280 = 0.05 to 0.1
  • Quenchers: these are most conveniently made up as concentrated stock solutions; examples of frequently used quenchers include:
    • 5 M NaI or 2.5 M KI solution containing 1 mM Na 2S 2O 3 to prevent formation of reactive I 3
    • 5 M CsCl (optical grade, Aldrich)
    • 8 M acrylamide (Electran grade from BDH; ɛ 295 = 0.236 liter/mol/cm)
    • 2.5 M succinimide (recrystallized from ethanol with activated charcoal treatment; ɛ 295 = 0.03 liter/mol/cm)
  • Additional reagents and equipment for recording a fluorescence spectrum (see protocol 1)
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Literature Cited

   Agashe, V.R., Shashtry, M.C.R., and Udgaonkar, J.B. 1995. Initial hydrophobic collapse in the folding of barstar. Nature 377:754‐757.
   Campbell, I.D. and Dwek, R.A. 1984. Biological Spectroscopy. Benjamin/Cummings, Menlo Park, Calif.
   Cardamone, M. and Puri, K. 1992. Spectrofluorimetric assessment of the surface hydrophobicity of proteins. Biochem. J. 282:589‐593.
   Christensen, H. and Pain, R.H. 1994. The contribution of the molten globule model. In Mechanisms of Protein Folding (R.H. Pain, ed.) pp. 55‐79. Oxford University Press, Oxford.
   Creamer, L.K. 1995. Effect of sodium dodecyl sulfate and palmitic acid on the equilibrium unfolding of bovine β‐lactoglobulin. Biochemistry 34:7170‐7176.
   Eftink, M.R. and Ghiron, C. 1981. Fluorescence quenching studies with proteins. Anal. Biochem. 114:199‐227.
   Freifelder, D. 1982. Physical Biochemistry: Applications to Biochemistry and Molecular Biology. Freeman, New York.
   Gruebele, M. 1999. The fast protein folding problem. Annu. Rev. Phys. Chem. 50:485‐516.
   Hlodan, R. and Pain, R.H. 1994. Tumour necrosis factor is in equilibrium with a trimeric molten globule at low pH. FEBS Lett. 343:256‐260.
   Ikeuchi, Y., Nakagawa, K., Endo, T., Suzuki, A., Hayashi, T., and Ito, T. 2001. Pressure‐induced denaturation of monomer β‐lactoglobulin is partially irreversible: Comparison of monomer form (highly acidic pH) with dimer form (neutral pH). J. Agric. Food Chem. 49:4052‐4059.
   Kuwajima, K. and Arai, M. 2000. The molten globule state: The physical picture and biological significance. In Mechanisms of Protein Folding (R.H. Pain, ed.) pp. 138‐174. Oxford University Press, Oxford.
   Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy. Plenum Press, New York.
   Losso, J.N., Kummer, A., Li‐Chan, E., and Nakai, S. 1993. Development of a particle concentration fluorescence immunoassay for the quantitative determination of IgG in bovine milk. J. Agric. Food Chem. 41:682‐686.
   Schmid, F.X. 1989. Spectral methods of characterizing protein conformation and conformational changes. In Protein Structure (T.E. Creighton, ed.) pp. 251‐285. IRL Press, Oxford.
   Varley, P.G., Dryden, D.T., and Pain, R.H. 1991. Resolution of the fluorescence of the buried tryptophan in yeast 3‐phosphoglycerate kinase using succinimide. Biochim. Biophys. Acta 1077:19‐24.
Key Reference
   Lakowicz, J.R. 1983. See above.
  An essential text, providing a thorough, but clear and readable description of the practice and interpretation of fluorescence spectroscopy, with particular reference to proteins.
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