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Practical High Performance Liquid Chromatography of Proteins and Peptides

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High Performance Liquid Chromatography in Plant Sciences

Part of the book series: Modern Methods of Plant Analysis ((MOLMETHPLANT,volume 5))

Abstract

Many biologically active proteins and peptides are present in the cytoplasm or on the cell membrane in rather small amounts, and for a long time researchers could only demonstrate their presence by bioassays of impure preparations or by immunoprecipitation and autoradiography. However, with the introduction and commercial availability of the highly sensitive gas-phase protein sequenator, structural studies of many of these polypeptides became feasible for an increasing number of biological research laboratories. These researchers are faced with the task of isolating specific proteins often present in very dilute solutions with complex mixtures of other proteins. Classical biochemical chromatographic techniques are hardly suitable for these subnanomole levels and in addition usually include lengthy and complex multistep procedures. Fortunately, high performance liquid chromatography (HPLC) allows fast micropreparative isolation of proteins and their peptide fragments. This technique has resulted in a spectacular improvement in speed, resolution, and sensitivity due to the use of stationary phases that consist of very small and uniform porous particles with high ligand densities. This allows better mass transfer, higher column efficiencies and higher capacities for polypeptides. Consequently, high performance separations can be obtained on small columns in a short time. However, high pressures are necessary to force the mobile phase through the column packings. Several modes of interaction between column supports and polypeptides exist and the chromatographic techniques are usually named after these mechanisms involved. Size exclusion (SE), ion-exchange (IEX) and hydrophobic interaction are by far the most well known and are the basis for SE-HPLC, IEX-HPLC and reversed phase (RP)-HPLC. In this review we will focus on these three techniques. It has to be mentioned, however, that in spite of the paucity of applications described in the literature, hydrophobic interaction, hydroxy apatite and affinity-matrix HPLC have also been used for polypeptide isolation.

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Authors
  1. P. Tempst
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  2. L. E. Hood
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  3. S. B. H. Kent
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Editors and Affiliations

  1. Goldberglein 7, D-8520, Erlangen, Germany

    Hans-Ferdinand Linskens

  2. Department of Biochemistry, Waite Agricultural Research Institute, University of Adelaide, 5064, Glen Osmond, S.A., Australia

    John F. Jackson

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© 1987 Springer-Verlag Berlin Heidelberg

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Tempst, P., Hood, L.E., Kent, S.B.H. (1987). Practical High Performance Liquid Chromatography of Proteins and Peptides. In: Linskens, HF., Jackson, J.F. (eds) High Performance Liquid Chromatography in Plant Sciences. Modern Methods of Plant Analysis, vol 5. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-82951-2_11

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  • DOI: https://doi.org/10.1007/978-3-642-82951-2_11

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