A new resorufin-based α-glucosidase assay for high-throughput screening
Section snippets
Reagents and chemicals
4MU-α-glc, NB–DNJ, a known inhibitor of GAA, and the buffer components were purchased from Sigma–Aldrich (St. Louis, MO, USA). GAA was obtained from residual solution after clinical infusions of Myozyme (Genzyme). The enzyme solution was mixed with 30% glycerol, and small aliquots were stored at −80 °C for up to 2 years.
The assay buffer was composed of 50 mM citric acid, 115 mM K2PO4, 110 mM KCl, 10 mM NaCl, 1 mM MgCl2, and 0.01% Tween 20 at pH 5.0. It was stored at 4 °C for up to 6 months. A solution
Results and discussion
On hydrolysis by GAA, fluorogenic res-α-glc forms two products: glucose and resorufin. Resorufin has a pKa of approximately 6.0 and emits red fluorescence at a peak of 590 nm (Fig. 1). This substrate has advantages over the existing substrate, 4MU-α-glc, which emits blue fluorescence at a peak of 440 nm, as explained below. The fluorescence enzyme assay is usually more sensitive than the chromogenic enzyme assay and is a better choice for assay miniaturization for HTS. However, a number of
Acknowledgments
This research was supported by the Molecular Libraries Initiative of the National Institutes of Health (NIH) Roadmap for Medical Research and the Intramural Research Programs of the National Human Genome Research Institute. The authors thank Paul Shinn for assistance with compound management.
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