Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10532554 | Analytical Biochemistry | 2013 | 8 Pages |
Abstract
Catalytic turnover of pyruvate to lactate by lactate dehydrogenase (LDH) is critical in maintaining an intracellular nicotinamide adenine dinucleotide (NAD+) pool for continuous fueling of the glycolytic pathway. In this article, we describe two label-free high-throughput assays (a kinetic assay detecting the intrinsic reduced nicotinamide adenine dinucleotide (NADH) fluorescence and a mass spectrometric assay monitoring the conversion of pyruvate to lactate) that were designed to effectively identify LDH inhibitors, characterize their different mechanisms of action, and minimize potential false positives from a small molecule compound library screen. Using a fluorescence kinetic image-based reader capable of detecting NADH fluorescence in the ultra-high-throughput screening (uHTS) work flow, the enzyme activity was measured as the rate of NADH conversion to NAD+. Interference with NADH fluorescence by library compounds was readily identified during the primary screen. The mass spectrometric assay quantitated the lactate and pyruvate levels simultaneously. The multiple reaction monitoring mass spectrometric method accurately detected each of the two small organic acid molecules in the reaction mixture. With robust Zâ² scores of more than 0.7, these two high-throughput assays for LDH are both label free and complementary to each other in the HTS workflow by monitoring the activities of the compounds on each half of the LDH redox reaction.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Erica VanderPorten, Lauren Frick, Rebecca Turincio, Peter Thana, William LaMarr, Yichin Liu,