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A Sensitive Fluorimetric Assay for Detection of Beta‐Secretase Using a Novel FRET Peptide Substrate
Author(s) -
Zhu Xudong,
Han Xing,
Lu Qing,
Meyer Rich,
Tong Xiaohe,
Rakhmanova Vera
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a19-b
Subject(s) - förster resonance energy transfer , fluorophore , chemistry , amyloid beta , substrate (aquarium) , enzyme , protease , fluorescence , peptide , ic50 , beta (programming language) , chromatography , biochemistry , in vitro , biology , ecology , physics , quantum mechanics , computer science , programming language
Beta‐secretase is a protease identified as a key enzyme in the deposition of beta‐amyloid plaques found in Alzheimer's disease. In order to facilitate high throughput screening of Alzheimer's disease drug candidates, we have developed a new Enzolyte™ 520 beta‐secretase assay kit, based on fluorescence resonance energy transfer (FRET), to replace less convenient HPLC‐based assays. The substrate is a nonapeptide labeled with the fluorophore Hilyte™ 488 and the quencher QXL™ 520. This assay showed good sensitivity, resulting in signal‐to‐background ratios of greater than 10 after 40 minutes and over 100 after 16 hr. The measured IC 50 values for known inhibitors were comparable to literature values. Since the formation of product can be continuously monitored, the assay may also be used to determine the kinetic parameters of the enzyme reaction.