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 (Fe^IIIXO) formation was established. This method showed that due to the conversion of Fe^II to Fe^III by H₂O₂ and subsequent formation of Fe^IIIXO complex halo in agar medium, the logarithm of H₂O₂ concentration from 5 to 160 µM was linearly correlated to the diameter of purplish red Fe^IIIXO halo. By extracting the LAAO-generated H₂O₂ concentration, the LAAO activity can be quantitatively determined. This Fe^IIIXO 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 Fe^IIIXO 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.