Skip to main content
SpringerLink
Log in

Polyol Additives Modulate the In Vitro Stability and Activity of Recombinant Human Phenylalanine Hydroxylase

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Phenylketonuria (PKU; OMIM 261600), the most common disorder of amino acid metabolism, is caused by a deficient activity of human phenylalanine hydroxylase (hPAH). Although the dietetic treatment has proven to be effective in preventing the psycho-motor impairment, much effort has been made to develop new therapeutic approaches. Enzyme replacement therapy with hPAH could be regarded as a potential form of PKU treatment if the reported in vitro hPAH instability could be overcome. In this study, we investigated the effect of different polyol compounds, e.g. glycerol, mannitol and PEG-6000 on the in vitro stability of purified hPAH produced in a heterologous prokaryotic expression system. The recombinant human enzyme was stored in the presence of the studied stabilizing agents at different temperatures (4 and −20 °C) during a 1-month period. Protein content, degradation products, specific activity, oligomeric profile and conformational characteristics were assessed during storage. The obtained results showed that the use of 50% glycerol or 10% mannitol, at −20 °C, protected the enzyme from loss of its enzymatic activity. The determined ΔG 0 and quenching parameters indicate the occurrence of conformational changes, which may be responsible for the observed increase in catalytic efficiency.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Lichter-Konecki, U., Hipke, C. M., & Konecki, D. S. (1999). Molecular Genetics and Metabolism, 67, 308–316.

    Article  CAS  Google Scholar 

  2. Moller, N., Meek, S., Bigelow, M., Andrews, J., & Nair, K. S. (2000). Proceedings of National Academy of Sciences USA, 97, 1242–1246.

    Article  CAS  Google Scholar 

  3. Scriver, C. R. (2007). Human Mutation, 28, 831–845.

    Article  CAS  Google Scholar 

  4. Surtees, R., & Blau, N. (2000). European Journal of Pediatrics, 159(Suppl 2), S109–S113.

    Article  CAS  Google Scholar 

  5. (1993). Arch Dis Child, 68, 426–427.

  6. (2001). Pediatrics, 108, 972–982.

  7. de Freitas, O., Izumi, C., Lara, M. G., & Greene, L. J. (1999). Nutrition Reviews, 57, 65–70.

    Article  Google Scholar 

  8. Widaman, K. F., & Azen, C. (2003). Pediatrics, 112, 1537–1543.

    Google Scholar 

  9. Kure, S., Hou, D. C., Ohura, T., Iwamoto, H., Suzuki, S., Sugiyama, N., et al. (1999). Journal of Pediatrics, 135, 375–378.

    Article  CAS  Google Scholar 

  10. Blau, N., & Erlandsen, H. (2004). Molecular Genetics and Metabolism, 82, 101–111.

    Article  CAS  Google Scholar 

  11. Sarkissian, C. N., Shao, Z., Blain, F., Peevers, R., Su, H., Heft, R., et al. (1999). Proceedings of National Academy of Sciences USA, 96, 2339–2344.

    Article  CAS  Google Scholar 

  12. Gamez, A., Sarkissian, C. N., Wang, L., Kim, W., Straub, M., Patch, M. G., et al. (2005). Molecular Therapeutic, 11, 986–989.

    Article  CAS  Google Scholar 

  13. Sarkissian, C. N., & Gamez, A. (2005). Molecular Genetics and Metabolism, 86(Suppl 1), S22–S26.

    Article  CAS  Google Scholar 

  14. Flatmark, T., & Stevens, R. C. (1999). Chemical Reviews, 99, 2137–2160.

    Article  CAS  Google Scholar 

  15. Thorolfsson, M., Ibarra-Molero, B., Fojan, P., Petersen, S. B., Sanchez-Ruiz, J. M., & Martinez, A. (2002). Biochemistry, 41, 7573–7585.

    Article  CAS  Google Scholar 

  16. Frokjaer, S., & Otzen, D. E. (2005). Nature Reviews Drug Discovery, 4, 298–306.

    Article  CAS  Google Scholar 

  17. Crommelin, D. J., Storm, G., Verrijk, R., de Leede, L., Jiskoot, W., & Hennink, W. E. (2003). International Journal of Pharmaceutics, 266, 3–16.

    Article  CAS  Google Scholar 

  18. Wang, W. (1999). International Journal of Pharmaceutics, 185, 129–188.

    Article  CAS  Google Scholar 

  19. Gamez, A., Wang, L., Straub, M., Patch, M. G., & Stevens, R. C. (2004). Molecular Therapeutic, 9, 124–129.

    Article  CAS  Google Scholar 

  20. Kwok, S. C., Ledley, F. D., DiLella, A. G., Robson, K. J., & Woo, S. L. (1985). Biochemistry, 24, 556–561.

    Article  CAS  Google Scholar 

  21. Leandro, P., Rivera, I., Lechner, M. C., de Almeida, I. T., & Konecki, D. (2000). Molecular Genetics and Metabolism, 69, 204–212.

    Article  CAS  Google Scholar 

  22. Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  23. Kaufman, S. (1987). Methods in Enzymology, 142, 3–17.

    Article  CAS  Google Scholar 

  24. Kleppe, R., Uhlemann, K., Knappskog, P. M., & Haavik, J. (1999). Journal of Biological Chemistry, 274, 33251–33258.

    Article  CAS  Google Scholar 

  25. Pace, C. N. (1986). Methods in Enzymology, 131, 266–280.

    Article  CAS  Google Scholar 

  26. Knappskog, P. M., & Haavik, J. (1995). Biochemistry, 34, 11790–11799.

    Article  CAS  Google Scholar 

  27. Lehrer, S. S. (1971). Biochemistry, 10, 3254–3263.

    Article  CAS  Google Scholar 

  28. Knappskog, P. M., Flatmark, T., Aarden, J. M., Haavik, J., & Martinez, A. (1996). European Journal of Biochemistry, 242, 813–821.

    Article  CAS  Google Scholar 

  29. Kaufman, S. (1993). Advances in Enzymology and Related Areas of Molecular Biology, 67, 77–264.

    Article  CAS  Google Scholar 

  30. Woo, S. L., Gillam, S. S., & Woolf, L. I. (1974). Biochemical Journal, 139, 741–749.

    CAS  Google Scholar 

  31. Chang, N., Kaufman, S., & Milstien, S. (1979). Journal of Biological Chemistry, 254, 2665–2668.

    CAS  Google Scholar 

  32. Wang, W. (2000). International journal of pharmaceutics, 203, 1–60.

    Article  CAS  Google Scholar 

  33. Powell, M. F. (1996). In R. Pearlman & Y. J. Wang (Eds.), Formulation, characterization and stability of protein drugs (pp. 1–140). New York: Plenum Press.

    Google Scholar 

  34. Shulgin, I. L., & Ruckenstein, E. (2006). Biophysical Chemistry, 120, 188–198.

    Article  CAS  Google Scholar 

  35. Pey, A. L., Desviat, L. R., Gamez, A., Ugarte, M., & Perez, B. (2003). Human Mutation, 21, 370–378.

    Article  CAS  Google Scholar 

  36. Bjorgo, E., de Carvalho, R. M., & Flatmark, T. (2001). European Journal of Biochemistry, 268, 997–1005.

    Article  CAS  Google Scholar 

  37. Shiman, R., Gray, D. W., & Pater, A. (1979). Journal of Biological Chemistry, 254, 11300–11306.

    CAS  Google Scholar 

  38. Andersen, O. A., Flatmark, T., & Hough, E. (2002). Journal of Molecular Biology, 320, 1095–1108.

    Article  CAS  Google Scholar 

  39. Stokka, A. J., Carvalho, R. N., Barroso, J. F., & Flatmark, T. (2004). Journal of Biological Chemistry, 279, 26571–26580.

    Article  CAS  Google Scholar 

  40. Timasheff, S. N. (1998). Advances in Protein Chemistry, 51, 355–432.

    Article  CAS  Google Scholar 

  41. Bolen, D. W., & Baskakov, I. V. (2001). Journal of Molecular Biology, 310, 955–963.

    Article  CAS  Google Scholar 

  42. Barteri, M., Gaudiano, M. C., & Santucci, R. (1996). Biochimica et Biophysica Acta, 1295, 51–58.

    Google Scholar 

  43. Lino, P. R., Almeida, A. J., de Tavares, A. I., & Leandro, P. (2009). FEBS Journal, 276, 323.

    Google Scholar 

Download references

Acknowledgements

This work was supported by Fundação para a Ciência e Tecnologia, Portugal, and FEDER: Project PTDC/QUI/64023/2006 and Grants SFRH/BD/10807/2002 (to Cátia Nascimento) and SFRH/BD/19024/2004 (to João Leandro).

Author information

Authors and Affiliations

  1. Metabolism and Genetics Group, iMed.UL, Faculdade Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisboa, Portugal

    Cátia Nascimento, João Leandro, Paulo Roque Lino, Luís Ramos, Isabel Tavares de Almeida & Paula Leandro

  2. Nanomedicine and Drug Delivery Systems Group, iMed.UL, Faculdade Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisboa, Portugal

    António José Almeida

Authors
  1. Cátia Nascimento
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. João Leandro
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paulo Roque Lino
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Luís Ramos
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. António José Almeida
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Isabel Tavares de Almeida
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Paula Leandro
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paula Leandro.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nascimento, C., Leandro, J., Lino, P.R. et al. Polyol Additives Modulate the In Vitro Stability and Activity of Recombinant Human Phenylalanine Hydroxylase. Appl Biochem Biotechnol 162, 192–207 (2010). https://doi.org/10.1007/s12010-009-8862-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-009-8862-y

Keywords

Search

Navigation