Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Studies on the iodination of aras protein and the detection ofras polymers

  • 18 Accesses

  • 6 Citations


Several methods for the iodination of recombinant v-H-ras protein were compared. The Iodobead method gave greates incorporation of radioactivity with minimal modification of theras protein. Upon treatment of theras protein with [125I] Nal and an Iodobead, radioactivity was initially incorporated into a 22 kDa species with a pl of 5.2, then predominantly into a 23 kDa species with a pl of 5.4. The specific activity of [125I]ras was 6×106 cpm/pmol totalras protein. Iondination did not alter the biological activity of theras protein as judged by its ability to bind GTPγS and induced maturation ofXenopus laevis oocytes. It is concluded that while iodination alters the apparent molecular weight and pI ofras, presumably by the oxidation of one or more classes of amino acids, this does not affect the biological function of the protein. Theras protein, radioactively-labelled with iodine using the Iodobead method, should be suitable for studies of protein-protein interactions involvingras. Treatment of iodinatedras with the chemical cross-linking agent disuccinimidyl suberate revealed the presence of several minor high molecular weight protein species. This result shows that, in a dilute solution of purifiedras protein, the monomeric form is in equilibrium with small amounts of polymeric forms.

This is a preview of subscription content, log in to check access.



Disuccinimidyl Suberate


Guanosine 5′-[γ-thio] triphosphate


Adenosine 5′[γ-thio] Triphosphate


  1. 1.

    Lowy DR, Willumsen BM: Function and regulation ofras. Ann Rev Biochem 62: 851–891, 1993

  2. 2.

    Egan SE, and Weinberg RA: The pathway to signal achievement. Nature 365: 781–783, 1993

  3. 3.

    Feig LA: The many roads that lead toras. Science 260: 767–768, 1993

  4. 4.

    Boguski MS, McCormick F: Proteins regulatingras and its relatives. Nature 366: 643–654, 1993

  5. 5.

    Moodie SA, Willumsen BM, Weber MJ and Wolfman A: Complexes ofras-GTP withraf-1 and mitogen-activated protein kinase kinase. Science 260: 1658–1661, 1993

  6. 6.

    Nur-E-Karmal MSA, Varga M, Maruta H: The GTPase-activating NF1 fragment of 91 amino acids reverses v-Ha-ras-induced malignant phenotype. J Biol Chem 268: 22331–22337, 1993

  7. 7.

    Downward J, Riehl R, Wu L, Weinberg RA: Identification of a nucleotide exchange-promoting activity for p21ras. Proc Natl Acad Sci USA 87: 5998–6002, 1990

  8. 8.

    Stagg BH, Temperley JW, Rochman H, Morley JS: Iodination and the biological activity of gastrin. Nature 228: 58–59, 1970

  9. 9.

    Heward CB, Yang YCS, Ormberg JF, Hadley ME, Hruby VJ: Effects of chloramine T and iodination on the biological activity of melanotropin. Hoppe-Seyler's Physiol Chem 360: 1851–1859, 1979

  10. 10.

    Santos E, Nebreda AR, Bryan T, Kempner ES: Oligomeric structure of p21ras proteins as determined by radiation inactivation. J Biol Chem 263: 9853–9858, 1988

  11. 11.

    Markwell MAK: A new solid-state reagent to iodinate proteins. Anal Biochem 125: 427–432, 1982

  12. 12.

    Tucker J, Sczakiel G, Feuerstein J, John J, Goody RS, Wittinghofer A: Expression of p21 proteins inEscherichia coli and stereochemistry of the nucleotide-binding site. EMBO J 5: 1351–1358, 1986

  13. 13.

    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275, 1951

  14. 14.

    Peterson GL: A simplification of the protein assay method of Lowryet al. which is more generally applicable. Analytical Biochem 83: 346–356, 1977

  15. 15.

    Janknecht R, de Martynoff G, Lou J, Hipskind RA, Nordheim A, Stunnenberg HG: Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci 88: 8972–8976, 1991

  16. 16.

    Shibuya EK, Polverino AJ, Chang E, Ruderman JV: Oncogenicras triggers the activation of 42-kDa mitogen-activated protein kinase in extracts of quiescentXenopus oocytes. Proc Natl Acad Sci 89: 9831–9835, 1992

  17. 17.

    Hunter WM, Greenwood FC: Preparation of iodine-132 labelled human growth hormone of high specific activity. Nature 194: 495–496, 1962

  18. 18.

    Harlow E, Lane D: Antibodies, a Laboratory Manual. Published by Cold Spring Harbour Laboratory, New York, pp 319–358, 1988

  19. 19.

    Fraker PJ, Speck JC: Protein and cell membrane iodinations with a sparingly soluble chloramide, 1,3,4,6-tetrachloro-3a, 6a-diphenylglycoluril. Biochem Biophys Res Commun 80: 849–857, 1978

  20. 20.

    Marchalonis JJ: An enzymic method for the trace iodinate proteins. Biochem J 113: 299–305, 1969

  21. 21.

    Morrison M, Bayes GS: Catalysis of iodination by lactoperoxidase. Biochemistry 9: 2995–3000, 1970

  22. 22.

    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680–685, 1970

  23. 23.

    Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979

  24. 24.

    Görg A, Postel W, Güther S: The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9: 531–546, 1988

  25. 25.

    Switzer RC, Merril CR, Shifrin S: A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Analytical Biochem 98: 231–237, 1979

  26. 26.

    Satoh T, Nakafuku M, Kaziro Y: Studies onras proteins. Catalytic properties of normal and activatedras proteins purified in the absence of protein denaturants. Biochim Biophys Acta 949: 97–109, 1988

  27. 27.

    Wu M, Kim R, Kim S: The use ofXenopus oocytes for the bioassay ofras. Methods: A companion to Methods in Enzymology 1: 315–318, 1990

  28. 28.

    McConahey PJ, Dixon FJ: Radioiodination of proteins by the use of the chloramine-T method. Methods Enzymol 70: 210–213, 1980

  29. 29.

    Morrison M, Schonbaum GR: Peroxidase-catalyzed halogenation. Ann Rev Biochem 45: 861–888, 1976

Download references

Author information

Correspondence to Greg J. Barritt.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chataway, T.K., Barritt, G.J. Studies on the iodination of aras protein and the detection ofras polymers. Mol Cell Biochem 137, 75–83 (1994). https://doi.org/10.1007/BF00926042

Download citation

Key words

  • ras
  • iodination
  • GTPγS binding
  • oocyte maturation
  • polymeric forms