Premium
Development of a Fluorescent NADPH Assay
Author(s) -
Diaz Elsie,
Schwartz Benjamin K.,
Patel Mehul P.,
Brandt Martin
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb51
Subject(s) - malic enzyme , enzyme , chemistry , biochemistry , cofactor , fluorescence , redox , absorbance , pentose phosphate pathway , oxidase test , chromatography , dehydrogenase , organic chemistry , glycolysis , physics , quantum mechanics
Many enzymes utilize NADPH as a cofactor, including those involved in redox matabolism. To date, assaying these enzymes has relied on changes in absorbance due to NADPH consumption; however monitoring changes in absorbance is limiting for higher throughput uses. This text describes the development of a high‐throughput fluorescence detection assay for NADPH utilizing enzymes, which couples the product (NADP+) to Malic Enzyme and Pyruvate Oxidase. The former catalyzes the reduction of NADP+ in the presence of malate and Mn2+ to produce pyruvate and NADPH, while the latter catalyzes the transformation of pyruvate and phosphate to acetyl phosphate, H202, and CO2. Horseradish peroxide (HRP) finally catalyzes the oxidation of amplex red to resorufin, which is fluoresencent. GlaxoSmithKline Pharmaceuticals.