Determinations of Polyamine Oxidase (PAO) Activity

Polyamine oxidase was extracted in 0.1 M phosphate buffer (pH 7.0), and the activity was measured as described previously (Chen et al., 2014). The reaction was initiated and incubated at 30°C for 30 min after addition of 20 μl of 20 mM Spd or Spd into reaction mixture (3 ml) that was consisted of 2.50 ml of 0.1 M phosphate buffer (pH 7.0), 0.1 ml of horseradish peroxidase (250 units), 0.2 ml of coloring solution (25 μl N, N-dimethylaniline and 10 mg 4-aminoantipyrine were dissolved in 100 ml of 0.1 M phosphate buffer, pH 7.0) and 0.2 ml enzyme extract or inactivated enzyme (by heating the enzyme for 20 min in a boiling water bath) as control. Absorbance at 550 nm was recorded. One unit of PAO activity was defined as the amount of enzyme required for catalyzing 1 μmol of Spd or Spm oxidation within 1 min.