文献类型：期刊文献

英文题名：Development of a new colorimetric assay for lipoxygenase activity

作者：Lu, Weiqiang[1];Zhao, Xue[1];Xu, Zhongyu[1];Dong, Ningning[1];Zou, Shien[2];Shen, Xu[1];Huang, Jin[1]

机构：[1]E China Univ Sci & Technol, Sch Pharm, Shanghai Key Lab New Drug Design, Shanghai Key Lab Chem Biol, Shanghai 200237, Peoples R China;[2]Fudan Univ, Obstet & Gynecol Hosp, Dept Gynecol, Shanghai 200011, Peoples R China

年份：2013

卷号：441

期号：2

起止页码：162

外文期刊名：ANALYTICAL BIOCHEMISTRY

收录：;EI(收录号：20201908624390);WOS:【SCI-EXPANDED(收录号:WOS:000324605400011)】；

基金：This work was supported by the 111 Project (grant B07023), the National Natural Science Foundation of China (grants 81102420 and 81200415), the Shanghai Committee of Science and Technology (grant 11DZ2260600), the Ph.D. Programs Foundation of the Ministry of Education of China (grant 20090071120020), and the Fundamental Research Funds for the Central Universities. The authors are appreciative of Colin D Funk (Department of Biochemistry, Queen's University, Canada) for the plasmids of pCDNA3-5-LOX, pCDNA3-p-12-LOX, and pCDNA3-15-LOX-1. The plasmid of stable 5-LOX was kindly provided by Marcia E. Newcomer (Department of Biological Sciences, Louisiana State University).

语种：英文

外文关键词：Lipoxygenase; Colorimetric assay; Ferrithiocyanate; Lipid hydroperoxides; Inhibitors

摘要：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 480 nm. Because 5-LOX can be stimulated by many cofactors, the effects of its cofactors (Ca2+, ATP, dithiothreitol, glutathione, L-alpha-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. (C) 2013 Elsevier Inc. All rights reserved.