values that were less than 30 mM. The redox absorbance assay has been used to qualitatively determine the redox mechanisms of inhibitors. Although the method is rapid and easy to handle, it has several shortcomings for Benzenesulfonamide,N-(4-ethylphenyl)-3-(hydroxymethyl)-N-(2-methylpropyl)-4-[(tetrahydro-2H-pyran-4-yl)methoxy]- practical use. To overcome these limitations, we introduced a fluorescence-based assay in this study. In the redox absorbance assay, the maximum absorbance change was very small, from 2.338 to 2.296. Although the molar extinction coefficient of 13-HpODE is solution can give a maximum absorbance change of 0.23, the practical absorbance change was much lower in studies conducted by others and us. One reason for the small absorbance change is that 13 -HODE, a product of consumption, also exhibits absorbance at the same wavelength. Therefore, degradation of the peroxide into its corresponding alcohol cannot be reflected by measuring absorbance. In addition, any decreases in absorbance were minimized by the elevated levels exhibited in the presence of DMSO. Reaction components, including the enzyme lysate, buffer components, DMSO, and inhibitor, appeared to contribute to the higher absorbance values at 234 nm. Endpoint measurements could not be made because of the large variation in starting absorbance values. All of these factors suggest that the absorbance assay is inaccurate and can only be used for kinetic purposes. On the contrary, the fluorescence signal of H2DCFDA was not affected by the reaction components and could I-BRD9 solely reflect the amount of remaining peroxide. The signal window ranged from as low as 300 to as high as 6000 at the same concentrations of inhibitors that were used in the absorbance assay. The possibility of signal interference seems rare, as it would occur only when the inhibitor fluoresces at the same wavelength as that of DCF. Fortunately, this would be easily recognizable by the abnormal shape of the dose-response curve. The redox mechanisms of a few inhibitors were ambiguous from the absorbance assay. This was partly due to the increase in negative signal in the absence of redox activity. The signal increase was also reported by others, and may be a result of an unknown reaction in the mixture. Because crude cell lysates
