title: A highly sensitive method for quantitative determination of L-amino acid oxidase activity based on the visualization of ferric-xylenol orange formation.
abstract: 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(III)XO) formation was established. This method showed that due to the conversion of Fe(II) to Fe(III) by H2O2 and subsequent formation of Fe(III)XO 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(III)XO halo. By extracting the LAAO-generated H2O2 concentration, the LAAO activity can be quantitatively determined. This Fe(III)XO 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(III)XO 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.
authors.affiliation: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Zhiliang
authors.initials: Z
authors.lastname: Yu
authors.affiliation: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Ju
authors.initials: J
authors.lastname: Wang
authors.affiliation: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhou
authors.affiliation: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Chuntian
authors.initials: C
authors.lastname: Zhao
authors.affiliation: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Juanping
authors.initials: J
authors.lastname: Qiu
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D005290:Ferric Compounds / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D050830:L-Amino Acid Oxidase / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism*; D031161:Pseudoalteromonas / Q000201:enzymology*; D012015:Reference Standards; D012996:Solutions; D013379:Substrate Specificity; D013454:Sulfoxides / Q000378:metabolism*
chemical_list: D005290:Ferric Compounds; D010636:Phenols; D012996:Solutions; D013454:Sulfoxides; D006861:Hydrogen Peroxide; D050830:L-Amino Acid Oxidase; C016833:xylenol orange