thod measures the amount of MCE Chemical 103476-89-7 peroxide by its absorbance at 234 nm, and the fluorescence-based method measures it by analyzing its reaction with H2DCFDA. H2DCF-DA was pre-cleaved by 5-LO crude lysate in the reaction buffer for more than 10 minutes before the main reaction, as previously reported. In a 384-well plate, enzyme solution was distributed and mixed with inhibitor solution with various concentrations. 13-HpODE was added to each well to start the reaction. After 3 minutes, pre-cleaved H2DCF-DA dye was added and incubated for more than 10 minutes. The fluorescence signal was measured using a fluorometer at excitation and emission wavelengths respectively. All steps were carried out at room temperature. The redox absorbance assay was carried out as described. Specifically inhibitor solution was mixed with 13-HpODE solution. After 3 minutes of incubation at room temperature, the solution was transferred to a cuvette. The reaction started when enzyme solution was added to the peroxide-inhibitor mixture in the cuvette. The accuracy and efficacy of the redox absorbance assay were determined by testing the selected compounds. The absorbance of 13-HpODE was measured the values were recorded every second for 3 minutes. These high values were due to contributions from the buffer, compounds, enzyme solution, and 13-HpODE. End-point measurements were not available, because the absorbance changes were less than the variations of the starting absorbance values. In theory, because the extinction coefficient of HpODE contributes in absorbance. The variations of the starting absorbance values are a result of the different absorptivity of the inhibitors. The results for all inhibitors were normalized by subtracting the starting absorbance values of the respective inhibitors. As shown previously, redox compounds induced rapid decreases in absorbance at the beginning of the reaction, and the velocity slowly declined as the lipid peroxide was consumed, which is the typical pattern for redox inhibitors. One DPC-681 example was zileuton, which showed a clear redox pattern with a reduction in absorbance. CAY10606 also showed decreases in absorbance, albeit at much weaker signals compared with that of zileuton. Non-redox compounds and DMSO controls showed slight increases in absorbance over time. Caffeic acid displayed no changes in ab
