Oligomeric States of Proteins Determined by Size-Exclusion Chromatography Coupled With Light Scattering, Absorbance, and Refractive Index Detectors

  • Ewa Folta-Stogniew
Part of the Methods in Molecular Biology™ book series (MIMB, volume 328)


Size-exclusion chromatography (SEC), coupled with “on-line” static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection, provides a universal approach for determination of the molar mass and oligomeric state in solution of native proteins as well as glycosylated proteins or membrane proteins solubilized in non-ionic detergents. Such glycosylated proteins or protein-detergent complexes show anomalous behavior on SEC, thus presenting a challenge to determination of molar mass and oligomeric state in solution. In the SEC-UV/LS/RI approach, SEC serves solely as a fractionation step, while the responses from the three detectors are utilized to calculate the molar mass for the polypeptide portion of the native or modified protein. The amount of sugar, lipid, or detergent bound to the polypeptide chain can also be estimated from the SEC-UV/LS/RI analysis.

Key Words

Laser light scattering size-exclusion chromatography (SEC) molar mass oligomeric state glycoproteins detergent-solubilized membrane proteins 


  1. 1.
    Wyatt P. J. (1993) Light scattering and the absolute characterization of macromolecules. Analytica Chimica Acta 272, 1–40.CrossRefGoogle Scholar
  2. 2.
    Folta-Stogniew E. and Williams K. R. (1999) Determination of molecular masses of proteins in solution: implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. J. Biomol. Tech. 10, 51–63.PubMedGoogle Scholar
  3. 3.
    Takagi T. (1990) Application of low-angle laser light scattering detection in the field of biochemistry: review of recent progress. J. Chromatog. A 506, 409–416.CrossRefGoogle Scholar
  4. 4.
    Wen J., Arakawa T., and Philo J. S. (1996) Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal. Biochem. 240, 155–166.PubMedCrossRefGoogle Scholar
  5. 5.
    Mogridge J. (2004) Using light scattering to determine the stoichiometry of protein complexes. Meth. Mol. Biol. 261, 113–118.Google Scholar
  6. 6.
    Hayashi Y., Matsui H., and Takagi T. (1989) Membrane protein molecular weight determined by low-angle laser light-scattering photometry coupled with highperformance gel chromatography. Meth. Enzymol. 172, 514–528.PubMedCrossRefGoogle Scholar
  7. 7.
    Hayashi Y., Takagi T., Maezawa S., and Matsui H. (1983) Molecular weights of [alpha][beta]-protomeric and oligomeric units of soluble (Na+, K+)-ATPase determined by low-angle laser light scattering after high-performance gel chromatography. Biochim. Biophys. Acta 748, 153–167.PubMedCrossRefGoogle Scholar
  8. 8.
    Kendrick B. S., Kerwin B. A., Chang B. S., and Philo J. S. (2001) Online sizeexclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states. Anal. Biochem. 299, 136–146.PubMedCrossRefGoogle Scholar
  9. 9.
    Wei Y., Li H., and Fu D. (2004) Oligomeric state of the Escherichia coli metal transporter YiiP. J. Biol. Chem. 279, 39,251–39,259.PubMedCrossRefGoogle Scholar
  10. 10.
    Yernool D., Boudker O., Folta-Stogniew E., and Gouaux E. (2003) Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus. Biochemistry 42, 12,981–12,988.PubMedCrossRefGoogle Scholar
  11. 11.
    Pace C. N., Vajdos F., Fee L., Grimsley G., and Gray T. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423.PubMedCrossRefGoogle Scholar
  12. 12.
    Prochazka O. and KratochvÌl P. (1980) Light scattering in multicomponent solutions a general equation. J. Polymer Sci. 18, 2369–2377.Google Scholar
  13. 13.
    Song L., Hobaugh M. R., Shustak C., Cheley S., Bayley H., and Gouaux J. E. (1996) Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1865.PubMedCrossRefGoogle Scholar
  14. 14.
    Schirmer T., Keller T. A., Wang Y. F., and Rosenbusch J. P. (1995) Structural basis for sugar translocation through maltoporin channels at 3.1 A resolution. Science 267, 512–514.PubMedCrossRefGoogle Scholar
  15. 15.
    Giddings J. C. (1993) Field-flow fractionation: analysis of macromolecular, colloidal, and particulate materials. Science 260, 1456–1465.PubMedCrossRefGoogle Scholar
  16. 16.
    D’ambra A. J., Baugher J. E., Concannon P. E., Pon R. A., and Michon F. (1997) Direct and indirect methods for molar-mass analysis of fragments of the capsular polysaccharide of Haemophilus influenzae type b. Anal. Biochem. 250, 228–236.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Ewa Folta-Stogniew

There are no affiliations available

Personalised recommendations