Microisolation of Biologically Active Polypeptides by Reverse-Phase Liquid Chromatography

  • Petro E. Petrides
Part of the Biological Methods book series (BM)


The development of new, high-sensitivity methods in protein chemistry (microisolation, amino acid microanalysis, and microse-quencing) has permitted the characterization of a large number of previously unknown polypeptides with regulatory functions. After decades of research devoted to the metabolism of the individual cell, this new methodology has sparked investigation into the presence and functional significance of polypeptides that govern the communication between cells within the multicellular organism. The technology is presently developing in two directions: (1) initial studies were done with small peptides, then progressed to medium-size polypeptides, and are now more and more utilized for proteins, and (2) less and less material is becoming necessary for structural characterization by amino acid analysis and sequencing, i.e., whereas initially several nanomoles of purified material were necessary, picomolar amounts are now required for partial or complete structural characterization. Over the years, reverse-phase liquid chromatography (RPLC) has evolved as the dominant tool for the isolation and characterization of polypeptides. Recently, however, alternative modes of chromatography, such as high-performance ion exchange, gel filtration, and chromatofocusing techniques, have been further developed, thus becoming integral parts of isolation programs.


Organic Modifier Teflon Tubing Sampling Valve Slider Valve Organic Solvent Concentration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Petrides, P.E. and Shooter, E.M. (1986) Rapid isolation of the 7S-NGF complex and its subunits from murine submaxillary glands and saliva. J. Neurochem. 46, 721–725.PubMedCrossRefGoogle Scholar
  2. 2.
    Lewis, R. V., Fallon, A., Stein, S., Gibson, K. D., and Udenfriend, S. (1980) Supports for reverse phase HPLC of large proteins. Anal. Biochem. 104, 153–159.PubMedCrossRefGoogle Scholar
  3. 3.
    Pearson, J. D., Mahoney, W. C., Hermodson, M. A., and Regnier, F. E. (1981) Reversed phase supports for the resolution of large denatured protein fragments. J. Chromatogr. 207, 325–332.PubMedCrossRefGoogle Scholar
  4. 4.
    Pearson, J. and Regnier, F. E. (1981) First Int. Symp. on HPLC of Proteins and Peptides. (Regnier, F. E., Hearn, M., and Wehr, T., eds.), Abstr. 308, Washington, DC.Google Scholar
  5. 5.
    Meienhofer, J., Gabriel, T. F., Michalewski, J., and Li, C. H. (1970) Large-Scale Purification of Peptides and Proteins by High Performance Liquid Chromatography, in Peptides 1978 Proc. Fourteenth European Peptides Symposium (Siemion, L. T., Kuprystewski, G., eds.), Wroclaw University Press, Wroclaw, Poland, pp. 243–246.Google Scholar
  6. 6.
    Pearson, J. and Regnier, F. (1983) The influence of reversed-phase n-alkyl chain length on protein retention, resolution, and recovery: Implications for preparative HPLC. J. Liq. Chromatogr. 6, 497–502.CrossRefGoogle Scholar
  7. 7.
    O’Hare, M. J. and Nice, E. C. (1979) Hydrophobic HPLC of hormonal polypeptides and proteins on alkyl-silane bonded silica. J. Chromatogr. 171, 209–226.PubMedCrossRefGoogle Scholar
  8. 8.
    Rubinstein, M. (1979) Preparative high performance liquid partition chromatography of proteins. Anal. Biochem. 98, 1–7.PubMedCrossRefGoogle Scholar
  9. 9.
    Petrides, P. E. and Böhlen, P. (1980) The mitogenic activity of insulin: An intrinsic property of the molecule. Biochem. Biophys. Res. Comm. 95, 1138–1144.PubMedCrossRefGoogle Scholar
  10. 10.
    Petrides, P. E., Jones, R. T., and Böhlen, P. (1980) RPLC of proteins: The efficient separation of hemoglobin variants. Anal. Biochem. 105, 383–388.PubMedCrossRefGoogle Scholar
  11. 11.
    Rubinstein, M., Rubinstein, S., Familetti, P. C., Miller, R. S., Waldman, A. A., and Pestka, S. (1979) Human leukocyte interferon: Production, purification to homogeneity and initial characterization. Proc. Natl. Acad. Sci. USA 76, 640–644.PubMedCrossRefGoogle Scholar
  12. 12.
    Rubinstein, S., Stein, S., Gerber, L., and Udenfriend, S. (1977) Isolation and characterization of the opioid peptides from rat pituitary: Lipotropin. Proc. Natl. Acad. Sci. USA 74, 3052–3055.PubMedCrossRefGoogle Scholar
  13. 13.
    Rivier, J. (1978) Use of trialkyl ammonium phosphate buffers in reverse phase HPLC for high resolution and high recovery of peptides and proteins. J. Liq. Chromatogr. 1, 343–366.CrossRefGoogle Scholar
  14. 14.
    Bennett, H. P. J., Browne, C. A., and Solomon, S. (1981) The use of perfluorinated carboxylic acids in the reverse phase HPLC of peptides. J. Liq. Chromatogr. 3, 1353–1365.CrossRefGoogle Scholar
  15. 15.
    Petrides, P. E., Hosang, M., Shooter, E. M., Esch, F., and Böhlen, P. (1985) Isolation and characterization of human epidermal growth factor from milk. FEBS Lett. 187, 89–95.PubMedCrossRefGoogle Scholar
  16. 16.
    Titani, K., Sasagawa, T., Resing, K. and Walsh, K. A. (1982) A simple and rapid purification of commercial trypsin and chymotrypsin by reverse-phase high performance liquid chromatography. Anal. Biochem. 123, 408–413.PubMedCrossRefGoogle Scholar
  17. 17.
    Kirschenbaum, D. M. (1981) A compilation of amino acid analyses of proteins. XVI. Residues per molecule. Int. J. Biochem. 13, 637–653.PubMedCrossRefGoogle Scholar
  18. 18.
    Böhlen, P., Stein, S., Stone, J., and Udenfriend, S. (1975) Automatic monitoring of primary amines in preparative column effluents with fluorescamine. Anal. Biochem. 67, 438–444.PubMedCrossRefGoogle Scholar
  19. 19.
    Darling, T., Petrides, P. E., Beguin, P., Frey, P., Shooter, E. M., Selby, M, and Rutter, W. J. (1983) The biosynthesis and processing of proteins in the mouse 7S nerve growth factor complex. Cold Spring Harbour Symposia on Quantitative Biology. XLVIII, 427–439.Google Scholar
  20. 20.
    Udenfriend, S., Stein, S., Böhlen, P., Daimann, W., Leimgruber, W., and Weigele, M. (1972) Applications of fluorescamine, a new reagent for assay of amino acids, peptides, proteins and other primary amines in the picomole range. Science 178, 871–873.PubMedCrossRefGoogle Scholar
  21. 21.
    Friesen, H. J., Stein, S., Evinger, M., Familetti, P. C., Moschera, J., Meienhofer, J., Shively, J., and Pestka, S. (1981) Purification and molecular characterization of human fibroblast interferon. Arch. Biochem. Biophys. 206, 432–444.PubMedCrossRefGoogle Scholar
  22. 22.
    O’Hare, M. H., Capp, M. W., Nice, E. C., Cooke, N. H. C., and Archer, B. G. (1983) Factors influencing chromatography of proteins on short alkysilane-bonded large pore size silicas. Anal. Biochem. 126, 17–28.CrossRefGoogle Scholar
  23. 23.
    Marquardt, H. and Todaro, G. J. (1982) Human transforming growth factor. J. Biol. Chem. 257, 5220–5225.PubMedGoogle Scholar
  24. 24.
    Wolfe, R. A., Casey, J., Familetti, P. C., and Stein, S. (1984) Isolation of proteins from crude mixtures with silica and silica-based adsorbents. J. Chromatogr. 296, 277–284.PubMedCrossRefGoogle Scholar
  25. 25.
    Suelter, C. H. and DeLuca, M. (1983) How to prevent losses of protein by adsorption to glass and plastic. Anal. Biochem. 135, 112–119.PubMedCrossRefGoogle Scholar
  26. 26.
    Petrides, P. E., Böhlen, P., and Shively, J. E. (1984) Chemical characterization of the two forms of epidermal growth factor in saliva. Biochem. Biophys. Res. Comm. 125, 218–228.PubMedCrossRefGoogle Scholar
  27. 27.
    Petrides, P. E., Hintz, R. L., Böhlen, P., and Shively, J. E. (1986) An improved method for the purification of human insulin like growth factors I and II. Endocrinology 18.Google Scholar
  28. 28.
    Petrides, P. E. (1984) Separation of Small Proteins, in Handbook of HPLC for the Separation of Amino Acids, Peptides and Proteins. Vol. II (Hancock, B., ed.), CRC, Boca Raton., pp. 327–344.Google Scholar
  29. 29.
    Petrides, P. E. (in preparation) Microisolation of regulatory polypeptides from body fluids: Use of preparative reverse-phase liquid chromatography.Google Scholar
  30. 30.
    Petrides, P. E., Hosang, M., Shooter, E. M., and Böhlen, P. (1984) Transforming growth factors in human milk. Isolation and biological and chemical characterization. Int. Congress of Endocrinology, Quebec City, Canada. Abstr. 1757, p. 1139.Google Scholar

Copyright information

© The Humana Press Inc. 1986

Authors and Affiliations

  • Petro E. Petrides
    • 1
  1. 1.Munich University School of MedicineGrossharden, MunichWest Germany

Personalised recommendations