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


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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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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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).

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