A Highly Sensitive Method for Quantitative Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Ferric-Xylenol Orange Formation

L-amino acid oxidase (LAAO) has important biological roles in many organisms, thus attracting great attention from researchers to establish its detection methods. In this study, a new quantitative in-gel determination of LAAO activity based on ferric-xylenol orange (FeIIIXO) formation was established. This method showed that due to the conversion of FeII to FeIII by H2O2 and subsequent formation of FeIIIXO complex halo in agar medium, the logarithm of H2O2 concentration from 5 to 160 µM was linearly correlated to the diameter of purplish red FeIIIXO halo. By extracting the LAAO-generated H2O2 concentration, the LAAO activity can be quantitatively determined. This FeIIIXO agar assay is highly sensitive to detect H2O2 down to micromolar range. More importantly, it is easy to handle, cheap, reproducible, convenient and accurate. Coupled with SDS-PAGE, it can directly be used to determine the number and approximate molecular weight of LAAO in one assay. All these features make this in-gel FeIIIXO assay useful and convenient as a general procedure for following enzyme purification, assaying fractions from a column, or observing changes in activity resulting from enzyme modifications, hence endowing this method with broad applications.