Sedimentation analysis of SDS and albumin-SDS complexes

  • K. J. Tiefenbach
  • H. Durchschlag
  • R. Jaenicke
Biological Systems
Part of the Progress in Colloid & Polymer Science book series (PROGCOLLOID, volume 107)


Knowledge of the structure of protein-SDS complexes and the constituent free proteins and SDS micelles is important for our understanding of protein-detergent interactions and for the application and improvement of physicochemical techniques connected with the purification and characterization of proteins. Several reasons are responsible for present controversies regarding the size and structure of macrosolutes, e.g. presence of a variety of low- and high-molecular solutes, the fact that macrosolutes show heterogeneity and may exhibit equilibria that are strongly influenced by the environmental conditions, and the point that techniques available for their structural investigation refer to different global or local properties. Sedimentation velocity and equilibrium experiments of the detergent-protein complexes and their constituents were performed under a variety of experimental conditions, such as rotor speed, scanning wavelength, choice of the baseline, the solvent or additives, and the concentrations of the components. Monitoring the sedimentation profiles of micellar SDS is facilitated by labeling the detergent micelles using a fluorescent dye, and discriminating the species under analysis by scanning at specific wavelengths. Interpretation of the results is facilitated by monitoring absorption spectra at discrete radial distances in the centrifuge cells, in addition to spectra recording of mixtures and components outside the centrifuge. In order to estimate the amount of bound detergent, sedimentation data (sedimentation coefficients, particle weights and mass distributions) were complemented by size-exclusion chromatographic studies. In the case of nonreduced proteins about 0.4 g SDS/g protein are bound at low detergent concentrations, and about 0.8 to 1.2 g/g at elevated concentrations.

Key words

Sodium dodecyl sulfate protein-detergent complexes micelles analytical ultracentrifugation absorption spectroscopy size-exclusion chromatography 



ammonium chloride


amplification factor


analytical ultracentrifugation


bovine serum albumin


chymotrypsinogen A


critical micelle concentration


critical micelle temperature


dextran blue


high-speed sedimentation equilibrium








sodium phosphate buffer






polyacrylamide gel electrophoresis


sodium dodecyl sulfate


size-exclusion chromatography


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Helenius A, Simons K (1975) Biochim Biophys Acta 415:29–79PubMedGoogle Scholar
  2. 2.
    Israelachvili JN, Mitchell DJ, Ninham BW (1976) J Chem Soc Faraday Trans II 72:1525–1568CrossRefGoogle Scholar
  3. 3.
    Tanford C, Reynolds JA (1976) Biochim Biophys Acta 457:133–170PubMedGoogle Scholar
  4. 4.
    Helenius A, McCaslin DR, Fries E, Tanford C (1979) Meth Enzymol 56:734–749PubMedGoogle Scholar
  5. 5.
    Tanford C (1980) The Hydrophobic Effect: Formation of Micelles and Biological Membrances, 2nd ed. Wiley, New YorkGoogle Scholar
  6. 6.
    Birdi KS (1985) Progr Colloid Polym Sci 70:23–29Google Scholar
  7. 7.
    Hoffmann H, Ulbricht W (1986) In: Hinz H-J (ed) Thermodynamic Data for Biochemistry and Biotechnology. Springer, Berlin, pp 297–348Google Scholar
  8. 8.
    Luisi PL, Magid LJ (1986) CRC Crit Rev Biochem 20:409–474PubMedCrossRefGoogle Scholar
  9. 9.
    Israelachvili JN (1992) Intermolecular and Surface Forces, 2nd ed. Academic Press, LondonGoogle Scholar
  10. 10.
    Myers D (1991) Surfaces, Interfaces, and Colloids: Principles and Applications. VCH, WeinheimGoogle Scholar
  11. 11.
    Moroi Y (1992) Micelles: Theoretical and Applied Aspects. Plenum Press, New YorkGoogle Scholar
  12. 12.
    Tanford C (1972) J Mol Biol 67:59–74PubMedCrossRefGoogle Scholar
  13. 13.
    Creighton TE (1993) Proteins: Structures and Molecular Properties, 2nd ed. WH Freeman, New YorkGoogle Scholar
  14. 14.
    Pitt-Rivers R, Impiombato FSA (1968) Biochem J 109:825–830PubMedGoogle Scholar
  15. 15.
    Reynolds JA, Tanford C (1970) Proc Natl Acad Sci USA 66:1002–1007PubMedCrossRefGoogle Scholar
  16. 16.
    Reynolds JA, Tanford C (1970) J Biol Chem 245:5161–5165PubMedGoogle Scholar
  17. 17.
    Nelson CA (1971) J Biol Chem 246:3895–3901PubMedGoogle Scholar
  18. 18.
    Tanford C, Nozaki Y, Reynolds JA, Makino S (1974) Biochemistry 13:2369–2376PubMedCrossRefGoogle Scholar
  19. 19.
    Takagi T, Tsujii K, Shirahama K (1975) J Biochem 77:939–947PubMedGoogle Scholar
  20. 20.
    Maddy AH (1976) J Theor Biol 62:315–326PubMedCrossRefGoogle Scholar
  21. 21.
    Makino S (1979) Adv Biophys 12:131–184PubMedGoogle Scholar
  22. 22.
    Mukerjee P, Mysels KJ (1971) Critical Micelle Concentrations of Aqueous Surfactant Systems. Nat Stand Ref Data Ser, Nat Bur Stand (USA), NSRDS-NBS 36, Washington DCGoogle Scholar
  23. 23.
    De Vendittis E, Palumbo G, Parlato G, Bocchini V (1981) Anal Biochem 115:278–286PubMedCrossRefGoogle Scholar
  24. 24.
    Chattopadhyay A, London E (1984) Anal Biochem 139:408–412PubMedCrossRefGoogle Scholar
  25. 25.
    Brito RMM, Vaz WLC (1986) Anal Biochem 152:250–255PubMedCrossRefGoogle Scholar
  26. 26.
    Samsonoff C, Daily J, Almog R, Berns DS (1986) J Colloid Interface Sci 109:325–329CrossRefGoogle Scholar
  27. 27.
    Sjöberg B, Pap S, Kjems J (1987) Eur J Biochem 162:259–264PubMedCrossRefGoogle Scholar
  28. 28.
    Fish WW, Reynolds JA, Tanford C (1970) J Biol Chem 245:5166–5168PubMedGoogle Scholar
  29. 29.
    Nozaki Y, Schechter NM, Reynolds JA, Tanford C (1976) Biochemistry 15:3884–3890PubMedCrossRefGoogle Scholar
  30. 30.
    Takagi T, Miyake J, Nashima T (1980) Biochim Biophys Acta 626:5–14PubMedGoogle Scholar
  31. 31.
    Kameyama K, Nakae T, Takagi T (1982) Biochim Biophys Acta 706:19–26PubMedGoogle Scholar
  32. 32.
    Jones MN, Midgley PJW (1984) Biochem J 219:875–881PubMedGoogle Scholar
  33. 33.
    Davis A (1984) In: Venter JC, Harrison LC (eds) Molecular and Chemical Characterization of Membrane Receptors. Alan R Liss, New York, pp 161–178Google Scholar
  34. 34.
    Ibel K, May RP, Kirschner K, Szadkowski H, Mascher E, Lundahl P (1990) Eur J Biochem 190:311–318PubMedCrossRefGoogle Scholar
  35. 35.
    Durchschlag H, Christl P, Jaenicke R (1991) Progr Colloid Polym Sci 86:41–56Google Scholar
  36. 36.
    Hirai M, Kawai-Hirai R, Hirai T, Ueki T (1993) Eur J Biochem 215:55–61PubMedCrossRefGoogle Scholar
  37. 37.
    Ibel K, May RP, Sandberg M, Mascher E, Greijer E, Lundahl P (1994) Biophys Chem 53:77–84PubMedCrossRefGoogle Scholar
  38. 38.
    Durchschlag H, Binder S, Christl P, Jaenicke R (1994) Jorn Com Esp Deterg 25:407–422 and Anexo 26–27Google Scholar
  39. 39.
    Durchschlag H, Zipper P (1995) Jorn Com Esp Deterg 26:275–292Google Scholar
  40. 40.
    Durchschlag H, Weber R, Jaenicke R (1996) In: Proc 4th World Surfactants Congress, Vol. 1. AEPSAT, Barcelona, pp 519–534Google Scholar
  41. 41.
    Durchschlag H, Tiefenbach K-J, Jaenicke R (1997) Jorn Com Esp Deterg 27:185–196 and Anexo 35–36Google Scholar
  42. 42.
    Shirahama K, Tsujii K, Takagi T (1974) J Biochem 75:309–319PubMedGoogle Scholar
  43. 43.
    Mattice WL, Riser JM, Clark DS (1976) Biochemistry 15:4264–4272PubMedCrossRefGoogle Scholar
  44. 44.
    Lundahl P, Greijer E, Sandberg M, Cardell S, Eriksson K-O (1986) Biochim Biophys Acta 873:20–26Google Scholar
  45. 45.
    Muga A, Arrondo JLR, Bellon T, Sancho J, Bernabeu C (1993) Arch Biochem Biophys 300:451–457PubMedCrossRefGoogle Scholar
  46. 46.
    Peters T Jr (1985) Adv Prot Chem 37:161–245CrossRefGoogle Scholar
  47. 47.
    Chervenka CH (1973) A Manual of Methods for the Analytical Ultracentrifuge. Spinco Division of Beckman Instruments, Palo AltoGoogle Scholar
  48. 48.
    Harding SE, Rowe AJ, Horton JC (eds) (1992) Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge (UK)Google Scholar
  49. 49.
    Schuster TM, Laue TM (eds) (1994) Modern Analytical Ultracentrifugation. Birkhäuser, BostonGoogle Scholar
  50. 50.
    Yphantis DA (1964) Biochemistry 3:297–317PubMedCrossRefGoogle Scholar
  51. 51.
    Reynolds JA, Tanford C (1976) Proc Natl Acad Sci USA 73:4467–4470PubMedCrossRefGoogle Scholar
  52. 52.
    Durchschlag H, Jaenicke R (1983) Int J Biol Macromol 5:143–148CrossRefGoogle Scholar
  53. 53.
    Reynolds JA, McCaslin DR (1985) Meth Enzymol 117:41–53PubMedGoogle Scholar
  54. 54.
    Schubert D, Schuck P (1991) Progr Colloid Polym Sci 86:12–22CrossRefGoogle Scholar
  55. 55.
    Roxby RW (1992) In: Harding SE, Rowe AJ, Horton JC (eds) Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry. Cambridge (UK) pp 609–618Google Scholar
  56. 56.
    Durchschlag H (1986) In: Hinz H-J (ed) Thermodynamic Data for Biochemistry and Biotechnology. Springer, Berlin, pp 45–128Google Scholar
  57. 57.
    Becker R, Helenius A, Simons K (1975) Biochemistry 14:1835–1841PubMedCrossRefGoogle Scholar
  58. 58.
    Mysels KJ, Princen LH (1959) J Phys Chem 63:1696–1700CrossRefGoogle Scholar
  59. 59.
    Anacker EW, Rush RM, Johnson JS (1964) J Phys Chem 68:81–93CrossRefGoogle Scholar
  60. 60.
    Fochler C, Durchschlag H (1997) Progr Colloid Polym Sci, this volumeGoogle Scholar

Copyright information

© Dr. Dietrich Steinkopff Verlag GmbH & Co. KG 1997

Authors and Affiliations

  • K. J. Tiefenbach
    • 1
  • H. Durchschlag
    • 1
  • R. Jaenicke
    • 1
  1. 1.Institute of Biophysics and Physical BiochemistryUniversity of RegensburgRegensburgGermany

Personalised recommendations