Journal
ANALYTICA CHIMICA ACTA
Volume 649, Issue 2, Pages 246-251Publisher
ELSEVIER
DOI: 10.1016/j.aca.2009.07.032
Keywords
Boronic acid; Saccharide; Enzyme assay; Fluorescence; Viologen
Categories
Funding
- [bio04-10458]
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In-vitro fluorescent enzyme assays have been developed for sucrose phosphorylase (SPO) and phosphoglucomutase (PGM). These assays make use of a selective carbohydrate sensing system that detects the unlabeled enzymatic products fructose and glucose-6-phosphate. The system comprises 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt as the reporter unit and boronic acid appended viologens as selective receptors with working ranges from 70 mu M to 1.0 mM for fructose (SPO) and 190 mu M to 2.0 mM for glucose-6-phosphate (PGM). The change in fluorescence can be converted into product concentration, allowing initial reaction velocities and Michaelis-Menten kinetics to be calculated. The assays are also carried out in multiwell plate formats, making them suitable for high-throughput screening of enzyme inhibitors. Rapid PGM inhibition screening is demonstrated with EDTA and LiCl. The PGM assay can also be used for enzyme quantification with a detection limit of 50 ng mL(-1). (C) 2009 Elsevier B.V. All rights reserved.
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