A wounding-induced PPO from cowpea ( Vigna unguiculata) seedlings

Abstract

Polyphenol oxidases (PPO) are induced in cowpea plants by wounding. The highest activity levels were detected 48 h after this stimulus in both wounded and neighbor-to-wounded unifoliates of cowpea seedlings; the increase of activity was in the order of 13 to 15-fold, respectively, in comparison to control unifoliates. Multiple molecular forms of active PPO (Mrs 58, 73 and ≅220 kDa) were detected by partially denaturing SDS–PAGE. Wounding-induced cowpea PPO were extracted and purified through (NH 4) 2SO 4 precipitation and ion-exchange chromatography. The effects of substrate specificity, pH, thermal stability and sensitivity to various inhibitors – resorcinol, EDTA, sodium azide and tropolone – of partially purified soluble PPO were investigated. Purified wounding-induced cowpea PPO (wicPPO) showed the highest activities towards 4-methylcatechol ( K m = 9.86 mM, V max = 24.66 EU [Δ A min −1]) and catechol ( K m = 3.44 mM, V max = 6.64 EU [Δ A min −1]); no activity was observed towards l-tyrosine, under the assay conditions used. The optimum pH for wound-induced cowpea PPO was 6.0 with 4-methylcatechol as substrate. The enzyme was optimally activated by 10 mM SDS and was highly stable even after 5 min at 80 °C. The most effective inhibitor was tropolone, whereas addition of 10 mM of resorcinol, EDTA and sodium azide were able to reduce PPO activities by 40%, 15% and 100%, respectively.