While the oxidized forms of these co-factors are “dark” and absorb weakly in the UV range, their reduced counterparts both absorb 340 nm light with extinction coefficients of over 6000 M−1 cm−1 and fluoresce at 450 nm. For example, hydroxysteroid dehydrogenase-catalyzed oxidation of estradiol can be measured by monitoring the increase in fluorescence intensity associated with the conversion of NAD to NADH. Because the fluorescence generated RGFP966 ic50 from NADH/NADPH is not very strong, compound fluorescence will interfere with this mode of detection. Therefore,
it is recommended that these assays are performed in a kinetic mode to increase the S:B and reduce compound interference. Coupling to diaphorase in the presence of resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) yields formation of resorufin which fluoresces at 590 nm, a region where fluorescent interference by LMW compounds is minimized. While this red-shifted reporting system is significantly less susceptible to compound fluorescence interference, it represents a less direct method for quantifying
the reaction progress and as such is more cumbersome to optimize and has a more limited dynamic range. Recently, a luminescent assay for either NADH or NADPH based on the reduction of a pro-luciferin substrate has been made available (Promega). For redox enzymes that generate/consume H2O2, the Amplex Red coupled assay is especially appropriate. In this case, VE-822 concentration catalase and resazurin are added at the end of the primary enzymatic reaction and the concentration of find more H2O2 can be measured
via the generation of highly fluorescent resorufin product (from the resazurin oxidation by H2O2). For lipoxygenases, a chromogenic assay has been published which can be used in HTS ( Cho et al., 2006) – lipoxygenase-catalyzed oxidation of linoleic or arachidonic acid substrate leads to the formation of lipid a peroxide product. In acidic pH conditions, this product can oxidize ferrous ions added at the end of the enzymatic reaction and the ferric ions generated in turn bind tightly to Xylenol Orange (absorption maximum of 405 nm) to form an intensely-colored complex with absorption maximum of 565 nm. The final product is stable over several hours, thus allowing batch-mode screening. One concern with peroxide detection, or any redox-based detection, is that certain compounds such as pyrimidotriazinediones in the presence of strong reducing reagents such as DTT will undergo a redox-cycling reaction to generate H2O2 (see section “Common Assay Artifacts in Biochemical Enzyme Assays”) ( Bova et al., 2004). A facile assay to check compounds for redox cycling involves simply adding the compounds under consideration to a reaction containing Amplex Red and horse-radish peroxidase alone ( Johnston et al., 2008).