Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Label-free continuous enzyme assays with macrocycle-fluorescent dye complexes

Abstract

We introduce a new economic, convenient and general assay principle based on the reversible interaction of water-soluble macrocycles and fluorescent dyes. We show that amino acid decarboxylase activity can be continuously monitored by measuring changes in fluorescence, which result from the competition of the enzymatic product and the dye for forming a complex with a cucurbituril or calixarene macrocycle. The new assay provides a complementary method to the use of antibodies, radioactive markers and labeled substrates.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Assay principle, chemical structures of the macrocycles and complexation equilibria with fluorescent dyes.
Figure 2: Fluorescence titrations.
Figure 3: Continuous fluorescence enzyme assays for lysine decarboxylase (in 10 mM NH4OAc buffer at pH 6.0).

References

  1. Sordé, N., Das, G. & Matile, S. Enzyme screening with synthetic multifunctional pores: focus on biopolymers. Proc. Natl. Acad. Sci. USA 100, 11964–11969 (2003).

    Article  Google Scholar 

  2. Goddard, J.P. & Reymond, J.-L. Enzyme assays for high-throughput screening. Curr. Opin. Biotechnol. 15, 314–322 (2004).

    Article  CAS  Google Scholar 

  3. Tawfik, D.S., Green, B.S., Chap, R., Sela, M. & Eshhar, Z. CatELISA—a facile general route to catalytic antibodies. Proc. Natl. Acad. Sci. USA 90, 373–377 (1993).

    Article  CAS  Google Scholar 

  4. Koh, K.N., Araki, K., Ikeda, A., Otsuka, H. & Shinkai, S. Reinvestigation of calixarene-based artificial-signaling acetylcholine receptors useful in neutral aqueous (water/methanol) solution. J. Am. Chem. Soc. 118, 755–758 (1996).

    Article  CAS  Google Scholar 

  5. Wiskur, S.L., Ait-Haddou, H., Lavigne, J.J. & Anslyn, E.V. Teaching old indicators new tricks. Acc. Chem. Res. 34, 963–972 (2001).

    Article  CAS  Google Scholar 

  6. Zhang, T. & Anslyn, E.V. Using an indicator displacement assay to monitor glucose oxidase activity in blood serum. Org. Lett. 9, 1627–1629 (2007).

    Article  CAS  Google Scholar 

  7. Bakirci, H., Koner, A.L., Dickman, M.H., Kortz, U. & Nau, W.M. Dynamically self-assembling metalloenzyme models based on calixarenes. Angew. Chem. Int. Ed. 45, 7400–7404 (2006).

    Article  CAS  Google Scholar 

  8. Gerner, E.W. & Meyskens, F.L. Polyamines and cancer: old molecules, new understanding. Nat. Rev. Cancer 4, 781–792 (2004).

    Article  CAS  Google Scholar 

  9. Blethen, S.L., Boeker, E.A. & Snell, E.E. Arginine decarboxylase from Escherichia coli. J. Biol. Chem. 243, 1671–1677 (1968).

    CAS  PubMed  Google Scholar 

  10. Soda, K. & Moriguchi, M. Crystalline lysine decarboxylase. Biochem. Biophys. Res. Commun. 34, 34–39 (1969).

    Article  CAS  Google Scholar 

  11. Tanase, S., Guirard, B.M. & Snell, E.E. Purification and properties of a pyridoxal 5′-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15. J. Biol. Chem. 260, 6738–6746 (1985).

    CAS  PubMed  Google Scholar 

  12. Phan, A.P.H., Ngo, T.T. & Lenhoff, H.M. Spectrophotometric assay for lysine decarboxylase. Anal. Biochem. 120, 193–197 (1982).

    Article  CAS  Google Scholar 

  13. Lee, J.W., Samal, S., Selvapalam, N., Kim, H.-J. & Kim, K. Cucurbituril homologues and derivatives: new opportunities in supramolecular chemistry. Acc. Chem. Res. 36, 621–630 (2003).

    Article  CAS  Google Scholar 

  14. Koner, A.L. & Nau, W.M. Cucurbituril encapsulation of fluorescent dyes. Supramol. Chem. 19, 55–66 (2007).

    Article  CAS  Google Scholar 

  15. Torres, F.E. et al. Enthalpy arrays. Proc. Natl. Acad. Sci. USA 101, 9517–9522 (2004).

    Article  CAS  Google Scholar 

  16. Geymayer, P., Bahr, N. & Reymond, J.-L. A general fluorogenic assay for catalysis using antibody sensors. Chem. Eur. J. 5, 1006–1012 (1999).

    Article  CAS  Google Scholar 

  17. Das, G. & Matile, S. Substrate-independent transduction of chromophore-free organic and biomolecular transformations into color. Chem. Eur. J. 12, 2936–2944 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J.-L. Reymond, S. Matile and H.-J. Schneider for valuable comments, and acknowledge financial support within the graduate program “Nanomolecular Science” at Jacobs University and by the Fonds der Chemischen Industrie, Frankfurt/Main.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Werner M Nau.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Methods (PDF 1368 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hennig, A., Bakirci, H. & Nau, W. Label-free continuous enzyme assays with macrocycle-fluorescent dye complexes. Nat Methods 4, 629–632 (2007). https://doi.org/10.1038/nmeth1064

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth1064

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing