Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Jun;83(12):4268–4272. doi: 10.1073/pnas.83.12.4268

Role of conformational changes in the elution of proteins from reversed-phase HPLC columns.

G E Katzenstein, S A Vrona, R J Wechsler, B L Steadman, R V Lewis, C R Middaugh
PMCID: PMC323713  PMID: 3459173

Abstract

To test the hypothesis that conformational alterations might be involved in the elution of proteins from reversed-phase HPLC columns, the conformations of proteins bound onto a C-8 alkyl-bonded silica surface have been examined in the presence of increasing concentrations of the commonly employed eluent, 1-propanol. Using a combination of photoacoustic, diffuse reflectance deconvolution Fourier transform infrared and front face fluorescence spectroscopic techniques (to minimize interference from light scattering), the existence of surface-associated protein conformational changes induced by propanol is unequivocally demonstrated. The linear relationship found between the amount of propanol needed to elute proteins from C-8 columns and the midpoint of spectrally observed structural transitions is consistent with a role for conformational changes in the elution process.

Full text

PDF
4268

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arakawa T., Goddette D. The mechanism of helical transition of proteins by organic solvents. Arch Biochem Biophys. 1985 Jul;240(1):21–32. doi: 10.1016/0003-9861(85)90004-9. [DOI] [PubMed] [Google Scholar]
  2. Benedek K., Dong S., Karger B. L. Kinetics of unfolding of proteins on hydrophobic surfaces in reversed-phase liquid chromatography. J Chromatogr. 1984 Dec 28;317:227–243. doi: 10.1016/s0021-9673(01)91662-0. [DOI] [PubMed] [Google Scholar]
  3. Burstein E. A., Vedenkina N. S., Ivkova M. N. Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol. 1973 Oct;18(4):263–279. doi: 10.1111/j.1751-1097.1973.tb06422.x. [DOI] [PubMed] [Google Scholar]
  4. Chirgadze Y. N., Fedorov O. V., Trushina N. P. Estimation of amino acid residue side-chain absorption in the infrared spectra of protein solutions in heavy water. Biopolymers. 1975 Apr;14(4):679–694. doi: 10.1002/bip.1975.360140402. [DOI] [PubMed] [Google Scholar]
  5. Cohen S. A., Benedek K., Tapuhi Y., Ford J. C., Karger B. L. Conformational effects in the reversed-phase liquid chromatography of ribonuclease A. Anal Biochem. 1985 Jan;144(1):275–284. doi: 10.1016/0003-2697(85)90117-4. [DOI] [PubMed] [Google Scholar]
  6. Eisinger J., Flores J. Front-face fluorometry of liquid samples. Anal Biochem. 1979 Apr 1;94(1):15–21. doi: 10.1016/0003-2697(79)90783-8. [DOI] [PubMed] [Google Scholar]
  7. Finney J. L., Gellatly B. J., Golton I. C., Goodfellow J. Solvent effects and polar interactions in the structural stability and dynamics of globular proteins. Biophys J. 1980 Oct;32(1):17–33. doi: 10.1016/S0006-3495(80)84913-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Geng X., Regnier F. E. Retention model for proteins in reversed-phase liquid chromatography. J Chromatogr. 1984 Jul 27;296:15–30. doi: 10.1016/s0021-9673(01)96399-x. [DOI] [PubMed] [Google Scholar]
  9. Hearn M. T. Reversed-phase high-performance liquid chromatography. Methods Enzymol. 1984;104:190–212. doi: 10.1016/s0076-6879(84)04090-8. [DOI] [PubMed] [Google Scholar]
  10. Herskovits T. T., Gadegbeku B., Jaillet H. On the structural stability and solvent denaturation of proteins. I. Denaturation by the alcohols and glycols. J Biol Chem. 1970 May 25;245(10):2588–2598. [PubMed] [Google Scholar]
  11. Macritchie F. Proteins at interfaces. Adv Protein Chem. 1978;32:283–326. doi: 10.1016/s0065-3233(08)60577-x. [DOI] [PubMed] [Google Scholar]
  12. Meek J. L. Prediction of peptide retention times in high-pressure liquid chromatography on the basis of amino acid composition. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1632–1636. doi: 10.1073/pnas.77.3.1632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Privalov P. L. Stability of proteins. Proteins which do not present a single cooperative system. Adv Protein Chem. 1982;35:1–104. [PubMed] [Google Scholar]
  14. Privalov P. L. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241. doi: 10.1016/s0065-3233(08)60460-x. [DOI] [PubMed] [Google Scholar]
  15. Purcell J. M., Susi H. Solvent denaturation of proteins as observed by resolution-enhanced Fourier transform infrared spectroscopy. J Biochem Biophys Methods. 1984 Jul;9(3):193–199. doi: 10.1016/0165-022x(84)90024-1. [DOI] [PubMed] [Google Scholar]
  16. Rosencwaig A. Photoacoustic spectroscopy. Annu Rev Biophys Bioeng. 1980;9:31–54. doi: 10.1146/annurev.bb.09.060180.000335. [DOI] [PubMed] [Google Scholar]
  17. Sadler A. J., Horsch J. G., Lawson E. Q., Harmatz D., Brandau D. T., Middaugh C. R. Near-infrared photoacoustic spectroscopy of proteins. Anal Biochem. 1984 Apr;138(1):44–51. doi: 10.1016/0003-2697(84)90766-8. [DOI] [PubMed] [Google Scholar]
  18. Sadler A. J., Micanovic R., Katzenstein G. E., Lewis R. V., Middaugh C. R. Protein conformation and reversed-phase high-performance liquid chromatography. J Chromatogr. 1984 Dec 28;317:93–101. doi: 10.1016/s0021-9673(01)91650-4. [DOI] [PubMed] [Google Scholar]
  19. Susi H., Byler D. M. Protein structure by Fourier transform infrared spectroscopy: second derivative spectra. Biochem Biophys Res Commun. 1983 Aug 30;115(1):391–397. doi: 10.1016/0006-291x(83)91016-1. [DOI] [PubMed] [Google Scholar]
  20. Wu C. S., Yang J. T. Sequence-dependent conformations of short polypeptides in a hydrophobic environment. Mol Cell Biochem. 1981 Oct 30;40(2):109–122. doi: 10.1007/BF00224754. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES