کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1176844 961887 2006 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Application of the ferrous oxidation–xylenol orange assay for the screening of 5-lipoxygenase inhibitors
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Application of the ferrous oxidation–xylenol orange assay for the screening of 5-lipoxygenase inhibitors
چکیده انگلیسی

5-Lipoxygenase (5-LO) is the key enzyme involved in leukotriene synthesis and its improper regulation is implicated in several inflammatory diseases. A rapid and sensitive assay for 5-LO activity suitable for high-throughput format is not yet available. In this study, we examined whether the ferrous oxidation–xylenol orange (FOX) assay could be applicable for the high-throughput screening of 5-LO inhibitors. Using insect cell lysates overexpressing rat 5-LO, the effects of cofactors of 5-LO such as ATP, Ca2+, and l-α-phosphatidylcholine (PC) on the color development of FOX reagents were investigated. ATP quenched substantially color development by hydroperoxide, an intermediate of 5-LO reaction, and an optimum concentration of ATP with little interference was determined as 20 μM. Ethylenediaminetetraacetate (0.4 mM) also affected the complex formation with FOX reagents. On the other hand, neither Ca2+ nor PC influenced complex formation with FOX reagents. Under optimized assay conditions, zileuton, a 5-LO-specific inhibitor, exhibited inhibitory potency (IC50 values of 0.1–0.2 μM) similar to that determined by the conventional spectrophotometric assay. Taken together, this study shows that the FOX assay with some modifications can be employed for high-throughput assay format for the measurement of 5-LO activity at the stage of primary screening.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 351, Issue 1, 1 April 2006, Pages 62–68
نویسندگان
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