Book Description

This doctoral thesis describes two novel fluorescent methods for assaying enzymatic activity, which are referred to as nanosecond time-resolved fluorescence (Nano-TRF) assays and supramolecular tandem assays. The first method, developed in collaboration with Fa. Hoffmann-La Roche, introduces a novel fluorescent probe (Dbo) for enzyme assays, which combines several desirable properties. First, Dbo has an exceedingly long fluorescence lifetime, which allows the use of Nano-TRF detection to increase the robustness of an assay by suppressing background fluorescence, for example from library compounds in high-throughput screening (HTS). Second, Dbo is efficiently quenched by tryptophan and tyrosine, which allows single-label assays. And third, Dbo has a very small size and hydrophilicity compared to common aromatic hydrophobic fluorescent probes. It has been demonstrated that the combination of these properties affords a minimally invasive, yet very powerful approach to determine the activity of proteases, tyrosine kinases and phosphatases. The second method introduces the use of water-soluble macrocycles and fluorescent dyes for enzyme assays, which presents an economic, convenient, and general assay principle. The assay is based on the competition of dye versus substrate and product in the reversible formation of a complex with the macrocycle. The enzyme thus converts a weak competitor (substrate) into a strong competitor (product) or vice versa, which leads to a different fraction of fluorescent dye bound to the macrocycle. Depending on whether the macrocycle/dye-complex is more or less fluorescent than the free dye, an increase or a decrease in fluorescence results, which signals the enzymatic activity. The method was applied to amino acid decarboxylases and arginase, and the possibility to derive enzyme kinetic parameters and inhibition constants has been demonstrated. Furthermore, the assays were conceptualized by supporting simulation.