Navigating Side Reactions for Robust Colorimetric Detection of Galactose Oxidase Activity

Colorimetric assays are a rapid, scalable technique well suited to enzyme activity screening. However, side reactions or chromogenic reagent instability can result in false positives or false negatives that compromise the accuracy of such assays. Here, we identify three classes of compounds incompatible with the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) colorimetric assay for galactose oxidase activity. Dark green ABTS^·+ cationic radicals indicating enzyme activity can get quenched to yield colorless solutions or couple with substrates to form differently colored adducts, thus preventing accurate colorimetric measurements. These side reactions limit the utility of the ABTS assay and introduce uncertainty in the substrate scope to which it is applicable. We have investigated the underlying mechanisms behind these side reactions to conclude that free radical scavengers, phenols with electron-donating substituents, and β,γ-unsaturated aryl ketones are incompatible with the ABTS colorimetric assay. In search of a viable alternative, we developed an assay using 2,4-dinitrophenylhydrazine under neutral conditions with isopropyl alcohol as a solubilizing agent. The use of neutral conditions was found to be critical to avoid hydrolysis of hydrazone adducts, ensuring reproducible measurements. Our assay is compatible with free radical scavengers (<i>R</i>² = 0.98), phenols with electron-donating substituents (<i>R</i>² = 0.97), and β,γ-unsaturated aryl ketones (<i>R</i>² = 0.88). This modified assay enables galactose oxidase activity screening across a broader substrate scope, thus facilitating enzyme use for more practical applications.