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Development of a fluorescent intensity assay amenable for high-throughput screening for determining 15-lipoxygenase activity.
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2010 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 15, no 6, p. 671-9Article in journal (Refereed) Published
Abstract [en]

15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids to form a lipid hydroperoxide. The authors have developed an assay for the detection of lipid hydroperoxides formed by human 15-lipoxygenase (15-LO) in enzyme or cellular assays using either a 96-well or a 384-well format. The assays described take advantage of the ability of lipid hydroperoxides to oxidize nonfluorescent diphenyl-1-pyrenylphosphine (DPPP) to a fluorescent phosphine oxide. Oxidation of DPPP yields a fluorescent compound, which is not sensitive to temperature and is stable for more than 2 h. The assay is sensitive toward inhibition and robust with a Z' value of 0.79 and 0.4 in a 96- and 384-well format, respectively, and thus amenable for high-throughput screening. The utility of DPPP as a marker for 15-lipoxygenase activity was demonstrated with both enzyme- and cell-based assays for the identification of hits and to determine potency by IC(50) determinations.

Place, publisher, year, edition, pages
2010. Vol. 15, no 6, p. 671-9
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Biomedical Laboratory Science/Technology
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URN: urn:nbn:se:uu:diva-343651DOI: 10.1177/1087057110373383PubMedID: 20581078OAI: oai:DiVA.org:uu-343651DiVA, id: diva2:1186517
Available from: 2018-02-28 Created: 2018-02-28 Last updated: 2018-02-28

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