title: Allosteric indicator displacement enzyme assay for a cyanogenic glycoside.
abstract: Indicator displacement assays (IDAs) represent an elegant approach in supramolecular analytical chemistry. Herein, we report a chemical biosensor for the selective detection of the cyanogenic glycoside amygdalin in aqueous solution. The hybrid sensor consists of the enzyme β-glucosidase and a boronic acid appended viologen together with a fluorescent reporter dye. β-Glucosidase degrades the cyanogenic glycoside amygdalin into hydrogen cyanide, glucose, and benzaldehyde. Only the released cyanide binds at the allosteric site of the receptor (boronic acid) thereby inducing changes in the affinity of a formerly bound fluorescent indicator dye at the other side of the receptor. Thus, the sensing probe performs as allosteric indicator displacement assay (AIDA) for cyanide in water. Interference studies with inorganic anions and glucose revealed that cyanide is solely responsible for the change in the fluorescent signal. DFT calculations on a model compound revealed a 1:1 binding ratio of the boronic acid and cyanide ion. The fluorescent enzyme assay for β-glucosidase uses amygdalin as natural substrate and allows measuring Michaelis-Menten kinetics in microtiter plates. The allosteric indicator displacement assay (AIDA) probe can also be used to detect cyanide traces in commercial amygdalin samples.
authors.affiliation: Institute for Inorganic and Analytical Chemistry, Abbe Center of Photonics, Friedrich Schiller University Jena, Humboldtstrasse 8, 07743 Jena (Germany); Current address: Department of Chemistry, National Institute of Technology, Kurukshetra, Haryana 136119 (India).
authors.forename: D Amilan
authors.initials: DA
authors.lastname: Jose
authors.forename: Martin
authors.initials: M
authors.lastname: Elstner
authors.forename: Alexander
authors.initials: A
authors.lastname: Schiller
mesh_terms: D000494:Allosteric Regulation; D000678:Amygdalin / Q000737:chemistry / Q000378:metabolism*; D001897:Boronic Acids / Q000737:chemistry / Q000378:metabolism; D003486:Cyanides / Q000737:chemistry*; D057075:Enzyme Assays; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism*; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D013379:Substrate Specificity; D014755:Viologens / Q000737:chemistry / Q000378:metabolism; D001617:beta-Glucosidase / Q000378:metabolism*
chemical_list: D001897:Boronic Acids; D003486:Cyanides; D005456:Fluorescent Dyes; D006027:Glycosides; D007477:Ions; D014755:Viologens; D000678:Amygdalin; D001617:beta-Glucosidase
keywords: amygdalin; boronic acid; chemical biosensors; density functional calculations; fluorescence