A sensitive fluorimetric assay for pyruvate.
A sensitive and specific fluorimetric assay for the determination of pyruvate is reported here. This assay is based on the oxidation of pyruvate in the presence of pyruvate oxidase. Hydrogen peroxide generated by pyruvate oxidase reacts with nonfluorescent Amplex Red at a 1:1 stoichiometry to form the fluorescent product, resorufin. The assay is optimized with respect to pH of reaction buffer, enzyme concentration, dye concentration, and the time course. The usefulness of the assay is demonstrated by the accurate measurement of intracellular and extracellular pyruvate concentrations. The limit of detection of the assay is 5 nM.
Zhu
Aiping
A
Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, MI 48105, USA.
Romero
Roberto
R
Petty
Howard R
HR
D001681:Biological Assay / Q000379:methods*; D002138:Calibration; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D057230:Limit of Detection; D019289:Pyruvic Acid / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results
D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase