A rapid and sensitive assay method for measuring amine oxidase based on hydrogen peroxide-titanium complex formation

Hydrogenperoxide (H2O2) is an end product of diamine and polyamine oxidation by their respective oxidase enzymes. A new sensitive assay method is based on a H2O2-titanium (Ti) complex formation as an indicator of H2O2 production due to polyamine oxidation. The orange-yellow coloured H2O2-Ti complex was measured at 410 nm in a Shimadzu spectrophotometer. The assay conditions for maximum diamine oxidase (DAO) and polyamine oxidase (PAO) as standardized here using the hypocotyl tissues of Vigna catjang Endl. cv Pusa Barsati consisted of pH 7.4 (40 mM potassium phosphate buffer), 3 mM substrate (putrescine or spermine), 37 degrees C incubation temperature and 30 min incubation time in the presence of catechol (10(-2) M) used as an inhibitor of both peroxidase and catalase activity. The method described here was significantly more sensitive than the starch-iodide method [T.A. Smith, Biochem. Biophys. Res. Commun. 41 (1970) 1452-1456], which could be improved further if measured under the same assay conditions as described for the H2O2-Ti method. Sensitivity of the present method was tested by assaying DAO/PAO activity in auxin treated hypocotyls of Vigna and comparing it with the starch-iodide method in two other plant samples. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.