Development of Capillary Electrophoretic Method with Fluorescence Detection for Beta-Secretase Activity Assays with FRET-Labeled Substrate
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| Year of publication | 2018 |
| Type | Conference abstract |
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| Description | Alzheimer's disease (AD) represents a degenerative brain disorder that is characterized by a progressive loss of memory and other cognitive abilities seriously affecting a person's ability to carry out daily activities. Currently available medications temporarily improve accompanying symptoms; however, a cure to treat the disease itself has not been introduced yet. Recent studies suggest beta-secretase, an aspartic-acid protease playing a key role in the development of neurotoxic amyloid plagues in the patients' brain tissue, as a promising druggable target to slow down or even stop the progression of AD. Screenings of potential beta-secretase inhibitors are commonly performed by using systems based on Förster resonance energy transfer (FRET). The information gathered from these assays can be negatively affected by low solubility of fluorescently labeled substrates and non-specific interferences with measured signal leading to a risk of false positive/negative results and need of data confirmation by further tests though. For these reasons the main aim of the presented study was to develop a method for activity studies of beta-secretase based on the capillary electrophoresis with fluorescence detection. Separation of a reaction product increases the assay sensitivity and diminishes the risk of interactions with other sample components. Separation conditions were optimized using standards of the QXL® 520/ HiLyteTM Fluor 488 FRET substrate and HiLyteTM 488 reaction product obtained from commercially available SensoLyte® 520 beta-secretase assay kit. An Agilent 7100 CE system equipped with Picometrics Zetalif LEDIF detector was used to perform all analyses. In the final method, the reaction product labeled with HiLyteTM 488 dye was separated by application of 30 kV (positive polarity) inside the uncoated fused-silica capillary (45 cm effective length, 50 ?m id) thermostated at 25 °C. 45 mM SDS prepared in 65 mM tetraborate buffer (pH 9.2) was used as BGE. The analyte was monitored at excitation/emission = 490 nm/520 nm. The capillary was rinsed with 0.1 M NaOH for 1 min, deionized water for 1 min, and BGE for 2 min before every analysis. This procedure was sufficient to ensure good method repeatability despite sample containing the enzyme and substrate and products peptides was directly injected into the capillary. The optimized method was validated and applied for activity assays of beta-secretase. The results obtained were in a good agreement with literature and confirmed the practical applicability of the developed method. |
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