Enzyme assays with boronic acid appended bipyridinium salts.
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 microM to 1.0 mM for fructose (SPO) and 190 microM 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).
Vilozny
Boaz
B
Department of Chemistry and Biochemistry, University of California, 1156 High Street, Santa Cruz, CA 95064, USA.
Schiller
Alexander
A
Wessling
Ritchie A
RA
Singaram
Bakthan
B
D001190:Arylsulfonates / Q000737:chemistry; D001897:Boronic Acids / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D005632:Fructose / Q000032:analysis*; D019298:Glucose-6-Phosphate / Q000032:analysis*; D005964:Glucosyltransferases / Q000378:metabolism*; D007700:Kinetics; D010733:Phosphoglucomutase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014755:Viologens / Q000737:chemistry
D001190:Arylsulfonates; D001897:Boronic Acids; D014755:Viologens; D005632:Fructose; D019298:Glucose-6-Phosphate; D004492:Edetic Acid; D005964:Glucosyltransferases; C025901:sucrose phosphorylase; D010733:Phosphoglucomutase; C005047:pyranine