Development of a new colorimetric assay for lipoxygenase activity

Lipoxygenases (LOXs) are a family of non-heme iron-containing dioxygenases that catalyze the hydroperoxidation of lipids, containing a cis,cis-1,4-pentadiene structure. A rapid and reliable colorimetric assay for determination of the activity of three human functional lipoxygenase isoforms (5-lipoxy...

Full description

Saved in:
Bibliographic Details
Published in:Analytical biochemistry 2013-10, Vol.441 (2), p.162-168
Main Authors: Lu, Weiqiang, Zhao, Xue, Xu, Zhongyu, Dong, Ningning, Zou, Shien, Shen, Xu, Huang, Jin
Format: Article
Language:English
Subjects:
absorbance
adenosine triphosphate
calcium
color
Colorimetric assay
colorimetry
Colorimetry - economics
Colorimetry - methods
dithiothreitol
drugs
EDTA (chelating agent)
Enzyme Assays - economics
Enzyme Assays - methods
Ferrithiocyanate
glutathione
HEK293 Cells
Humans
hydroperoxides
Inhibitors
inhibitory concentration 50
iron
Lipid hydroperoxides
lipids
Lipoxygenase
Lipoxygenase - metabolism
Lipoxygenase Inhibitors - pharmacology
Protein Isoforms - metabolism
screening
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Lipoxygenases (LOXs) are a family of non-heme iron-containing dioxygenases that catalyze the hydroperoxidation of lipids, containing a cis,cis-1,4-pentadiene structure. A rapid and reliable colorimetric assay for determination of the activity of three human functional lipoxygenase isoforms (5-lipoxygenase, platelet 12-lipoxygenase, and 15-lipoxygenase-1) is developed in this article. In the new assay, LOX-derived lipid hydroperoxides oxidize the ferrous ion (Fe2+) to the ferric ion (Fe3+), the latter of which binds with thiocyanate (SCN–) to generate a red ferrithiocyanate (FTC) complex. The absorbance of the FTC complex can be easily measured at 480nm. Because 5-LOX can be stimulated by many cofactors, the effects of its cofactors (Ca2+, ATP, dithiothreitol, glutathione, l-α-phosphatidylcholine, and ethylenediaminetetraacetic acid) on the color development of the FTC complex are also determined. The assay is adaptive for purified LOXs and cell lysates containing active LOXs. We use the new colorimetric assay in a 96-well format to evaluate several well-known LOX inhibitors, the IC50 values of which are in good agreement with previously reported data. The reliability and reproducibility of the assay make it useful for in vitro screening for inhibitors of LOXs and, therefore, should accelerate drug discovery for clinical application.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2013.06.007