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

Nature Methods volume 4, pages 629–632 (2007)Cite this article

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.

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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).

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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.

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  1. Andreas Hennig and Hüseyin Bakirci: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, Bremen, D-28759, Germany

    Andreas Hennig, Hüseyin Bakirci & Werner M Nau

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  1. Andreas Hennig
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  2. Hüseyin Bakirci
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Correspondence to Werner M Nau.

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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

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