Abstract
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.
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References
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.
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.
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.
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.
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.
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.
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.
Rubinstein, M. (1979) Preparative high performance liquid partition chromatography of proteins. Anal. Biochem. 98, 1–7.
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.
Petrides, P. E., Jones, R. T., and Böhlen, P. (1980) RPLC of proteins: The efficient separation of hemoglobin variants. Anal. Biochem. 105, 383–388.
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.
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.
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.
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.
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.
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.
Kirschenbaum, D. M. (1981) A compilation of amino acid analyses of proteins. XVI. Residues per molecule. Int. J. Biochem. 13, 637–653.
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.
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.
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.
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.
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.
Marquardt, H. and Todaro, G. J. (1982) Human transforming growth factor. J. Biol. Chem. 257, 5220–5225.
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.
Suelter, C. H. and DeLuca, M. (1983) How to prevent losses of protein by adsorption to glass and plastic. Anal. Biochem. 135, 112–119.
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.
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.
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.
Petrides, P. E. (in preparation) Microisolation of regulatory polypeptides from body fluids: Use of preparative reverse-phase liquid chromatography.
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.
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Petrides, P.E. (1986). Microisolation of Biologically Active Polypeptides by Reverse-Phase Liquid Chromatography. In: Shively, J.E. (eds) Methods of Protein Microcharacterization. Biological Methods. Humana Press. https://doi.org/10.1007/978-1-59259-436-8_1
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DOI: https://doi.org/10.1007/978-1-59259-436-8_1
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