title: A bioluminescence assay for aldehyde dehydrogenase activity.
abstract: The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B=64) and Z'=0.75.
authors.affiliation: Promega Corp., Madison, WI 53711, USA. Sarah.Duellman@promega.com
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Duellman
authors.forename: Michael P
authors.initials: MP
authors.lastname: Valley
authors.forename: Vinayaka
authors.initials: V
authors.lastname: Kotraiah
authors.forename: Jolanta
authors.initials: J
authors.lastname: Vidugiriene
authors.forename: Wenhui
authors.initials: W
authors.lastname: Zhou
authors.forename: Laurent
authors.initials: L
authors.lastname: Bernad
authors.forename: Jean
authors.initials: J
authors.lastname: Osterman
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Kimball
authors.forename: Poncho
authors.initials: P
authors.lastname: Meisenheimer
authors.forename: James J
authors.initials: JJ
authors.lastname: Cali
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000032:analysis* / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D008163:Luminescent Measurements; D015394:Molecular Structure
chemical_list: D000444:Aldehyde Dehydrogenase