A fluorescence-based assay for measuring the redox potential of 5-lipoxygenase inhibitors.
The activities and side effects of 5-lipoxygenase (5-LO) inhibitors can be predicted by identifying their redox mechanisms. In this study, we developed a fluorescence-based method to measure the redox potential of 5-LO inhibitors and compared it to the conventional, absorbance-based method. After the pseudo-peroxidase reaction, the amount of remaining lipid peroxide was quantified using the H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) fluorescence dye. Our method showed large signal windows and provided comparable redox potential values. Importantly, the redox mechanisms of known inhibitors were accurately measured with the fluorescence assay, whereas the conventional, absorbance-based method showed contradictory results. Our findings suggest that our developed method is a better alternative for classifying the redox potential of 5-LO inhibitors, and the fluorescence assay can be effectively used to study the mechanisms of action that are related to redox cycling.
Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Korea.
Sangchul
S
Lee
Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Korea.
Youngsam
Y
Park
Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Korea.
Junghwan
J
Kim
Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Korea.
Sung-Jun
SJ
Han
D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry*; D001681:Biological Assay / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins / Q000032:analysis; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D008054:Lipid Peroxides / Q000032:analysis; D016859:Lipoxygenase Inhibitors / Q000032:analysis* / Q000737:chemistry*; D010084:Oxidation-Reduction
C110400:2',7'-dichlorodihydrofluorescein diacetate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; D001094:Arachidonate 5-Lipoxygenase