Polyamines pp 207-216 | Cite as

Assay of Deoxyhypusine Hydroxylase Activity

  • Jong Hwan Park
  • Edith C. Wolff
  • Myung Hee Park
Part of the Methods in Molecular Biology book series (MIMB, volume 720)


The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains an unusual amino acid, hypusine [N ε-(4-amino-2-hydroxybutyl)-lysine]. eIF5A and its hypusine/deoxyhypusine modification are vital for eukaryotic cell proliferation. Hypusine is formed posttranslationally by two enzymatic steps catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. Deoxyhypusine hydroxylase catalyzes a stereo-specific hydroxylation of the deoxyhypusine residue in the eIF5A intermediate protein, eIF5A(Dhp). The enzyme is totally specific for this protein and does not act on short peptides (<50 amino acids). The assay measures the conversion of the radiolabeled deoxyhypusine residue to a hypusine residue in eIF5A. Optimum conditions for the reaction and two detection methods for the product, hypusine-containing eIF5A, are described in this chapter. The first, and most reliable, method is the measurement of the amount of [3H]hypusine in the protein hydrolysate after its separation from [3H]deoxyhypusine, by ion exchange chromatography. This method does require specialized equipment. The second method is based on counting the total TCA soluble radioactivity after sodium periodate oxidation of the reaction mixture, since the radiolabeled 4-amino-2-hydroxy butyl moiety of the hypusine residue is cleaved and is released from protein as radiolabeled β-propionaldehyde and formaldehyde by periodate oxidation.

Key words

Polyamine Spermidine Deoxyhypusine hydroxylase Deoxyhypusine Hypusine Monooxygenase eIF5A Posttranslational modification Protein hydroxylation 



This research was supported by the Intramural Research Program of the NIDCR, National Institutes of Health.


  1. 1.
    Park MH (2006) The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF5A). J Biochem 139:161–169PubMedCrossRefGoogle Scholar
  2. 2.
    Abbruzzese A, Park MH, Folk JE (1986) Deoxyhypusine hydroxylase from rat testis. Partial purification and characterization. J Biol Chem 261:3085–3089PubMedGoogle Scholar
  3. 3.
    Park JH, Aravind L, Wolff EC, Kaevel J, Kim YS, Park MH (2006) Molecular cloning, expression, and structural prediction of deoxyhypusine hydroxylase: a HEAT-repeat-containing metalloenzyme. Proc Natl Acad Sci USA 103:51–56PubMedCrossRefGoogle Scholar
  4. 4.
    Kang KR, Kim YS, Wolff EC, Park MH (2007) Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A. J Biol Chem 282:8300–8308PubMedCrossRefGoogle Scholar
  5. 5.
    Jakus J, Wolff EC, Park MH, Folk JE (1993) Features of the spermidine-binding site of deoxyhypusine synthase as derived from inhibition studies. Effective inhibition by bis- and mono- guanylated diamines and polyamines. J Biol Chem 268:13151–13159PubMedGoogle Scholar
  6. 6.
    Park MH, Cooper HL, Folk JE (1982) The biosynthesis of protein-bound hypusine (Nε-(4-amino-2-hydroxybutyl)lysine). Lysine as the amino acid precursor and the intermediate role of deoxyhypusine (Nε-(4-aminobutyl)lysine). J Biol Chem 257:7217–7222PubMedGoogle Scholar
  7. 7.
    Vu VV, Emerson JP, Martinho M, Kim YS, Munck E, Park MH, Que L Jr (2009) Human deoxyhypusine hydroxylase, an enzyme involved in regulating cell growth, activates O2 with a nonheme diiron center. Proc Natl Acad Sci USA 106:14814–14819PubMedCrossRefGoogle Scholar
  8. 8.
    Smit-McBride Z, Dever TE, Hershey JW, Merrick WC (1989) Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein. J Biol Chem 264:1578–1583PubMedGoogle Scholar
  9. 9.
    Joe YA, Wolff EC, Park MH (1995) Cloning and expression of human deoxyhypusine synthase cDNA. Struct function studies recombinant enzyme mutant proteins. J Biol Chem 270:22386–22392PubMedCrossRefGoogle Scholar
  10. 10.
    Smit-McBride Z, Schnier J, Kaufman RJ, Hershey JW (1989) Protein synthesis initiation factor eIF-4D. Funct comparison native unhypusinated forms protein. J Biol Chem 264:18527–18530PubMedGoogle Scholar
  11. 11.
    Joe YA, Park MH (1994) Structural features of the eIF-5A precursor required for posttranslational synthesis of deoxyhypusine. J Biol Chem 269:25916–25921PubMedGoogle Scholar
  12. 12.
    Park JH, Wolff EC, Folk JE, Park MH (2003) Reversal of the deoxyhypusine synthesis reaction. Generation spermidine or homospermidine deoxyhypusine by deoxyhypusine synthase. J Biol Chem 278:32683–32691PubMedCrossRefGoogle Scholar
  13. 13.
    Park MH, Cooper HL, Folk JE (1983) Chromatographic identification of hypusine (N  e-(4-amino-2-hydroxyl)lysine) and deoxyhypusine (Ne-(4-aminobutyl)lysine). Meth Enzymol 94:458–462CrossRefGoogle Scholar
  14. 14.
    Folk JE, Park MH, Chung SI, Schrode J, Lester EP, Cooper HL (1980) Polyamines as physiological substrates for transglutaminases. J Biol Chem 255:3695–3700PubMedGoogle Scholar
  15. 15.
    Park MH, Cooper HL, Folk JE (1981) Identification of hypusine, an unusual amino acid, in a protein from human lymphocytes and of spermidine as its biosynthetic precursor. Proc Natl Acad Sci USA 78:2869–2873PubMedCrossRefGoogle Scholar
  16. 16.
    Abbruzzese A, Park MH, Folk JE (1986) Indirect assays for deoxyhypusine hydroxylase using dual-label ratio changes and oxidative release of radioactivity. Anal Biochem 154:664–670PubMedCrossRefGoogle Scholar
  17. 17.
    Csonga R, Ettmayer P, Auer M, Eckerskorn C, Eder J, Klier H (1996) Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay. FEBS Lett 380:209–214PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Jong Hwan Park
    • 1
  • Edith C. Wolff
    • 1
  • Myung Hee Park
    • 1
  1. 1.The Oral and Pharyngeal Cancer BranchNIDCR, National Institutes of HealthBethesdaUSA

Personalised recommendations