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Jacobs University scientists under the lead of Werner Nau, Professor of Chemistry, developed a novel, highly efficient, economic and fast general method for measuring enzyme activity. The method is based on the reversible interaction of large circular molecules, so-called macrocycles, and fluorescent dyes. Used as simple additives to the enzyme reaction these “molecular spies” allow the direct observation of the whole reaction kinetics. Moreover, this method, which is published in the current advance online issue of Nature Methods (doi:10.1038/nmeth1064), requires significantly less preparational optimizing effort than customary assay methods.
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Jul
03, 2007]
The development of assays to detect the conversion of a substrate to a product is of fundamental importance for screening libraries of potential drugs and inhibitors, genetically engineered enzymes, and catalytic antibodies.
The new concept for enzymatic activity assays now presented by Werner Nau and his co-workers uses macrocyclic ‘receptor molecules’, such as cucurbiturils or calixarenes, and fluorescent dyes. The underlying assay principle is based on the self-assembly of a host-guest complex (the ‘reporter pair’) between the fluorescent dye and the macrocycle, where the dye is embedded in the cavity of the hollow macrocycle molecule. The macrocycle is specifically chosen so that it binds the enzyme substrate weakly but the product strongly. The substrate therefore does not displace the dye from the complex (weak
competitor) and remains in its uncomplexed form in solution, where it is enzymatically converted to the product, a strong competitor. With increasing concentration during the enzymatic reaction the product then gradually displaces the fluorescent dye from the macrocycle. Depending on whether the macrocyclic dye complex is more or less strongly fluorescent than the uncomplexed dye, the reaction can be directly spectroscopically followed through a fluorescence decrease (‘switch-off ’) or enhancement (‘switch-on’).
By applying this new assay principle, the activity of amino acid decarboxylases were monitored with unprecedented simplicity. Decarboxylases have a key role in tumor growth and inflammatory processes, which renders them important targets for drug discovery. Their enzymatic reactions have been previously followed by laborious manometric, radiometric, or multistep colorimetric methods, which are unsuitable for high-throughput screening.
Werner Nau about the advantages of the new method: “Our ‘molecular spies’ are commercially available and can usually be applied for whole enzyme classes. Other methods usually require optimization for each specific enzymatic reaction. Moreover, the cost efficiency of the new method is tremendous, easily more than 90 %, as it saves many time- and cost-intensive steps necessary for customary methods.”
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