铜铁试剂对菜青虫多酚氧化酶的抑制作用

Inhibitory effect of cupferron on the activity of polyphenoloxidase from Pieris rapae(L.) (Lepidoptera: Pieridae)

以菜青虫 Pieris rapae (L.) 为试材, 冰浴匀浆, 4℃下 6 403 ×g 离心, 取上清液, 经 35%饱和度 (NH4)2SO4 沉淀,Sephadex G-100凝胶过滤柱层析等分离步骤, 获得部分纯化的菜青虫多酚氧化酶制剂。研究不同浓度铜离子及铜铁试剂对该酶的影响, 结果表明: Cu2+在0~0.100 mmol/L范围内对酶活力表现激活作用; 浓度大于0.125 mmol/L时表现抑制作用, 其IC50为0.651±0.022 mmol/L; 铜铁试剂对该酶抑制作用的IC50为0.100±0.012 mmol/L。抑制动力学研究结果表明: 铜铁试剂对该酶表现为可逆抑制效应, 为竞争性抑制类型, 其抑制常数Ki 为0.076±0.013 mmol/L。

The kinetic properties of polyphenoloxidase (PPO) from Pieris rapae (L.) were studied after the enzyme was partially purified by 35% saturated (NH 4) 2SO 4 and chromatographed on a Sephadex G 100 gel filtration. Meanwhile, the effects of PPO on the oxidation of L DOPA by adding copper ion or cupferron were also studied. The results showed that the activity of PPO was enhanced by Cu^(2+) in the concentration of 0-0.100 mmol/L; however, the activity was inhibited by the same ion when the concentration was over 0.125 mmol/L, and the IC (50) was estimated to be 0.651±0.022 mmol/L. The results also indicated that the reaction of cupferron with the PPO is a reversible course with remaining enzyme activity, the inhibitory mechanism belongs to be the competitive type and the IC (50) was estimated to be 0.100±0.012 mmol/L. Furthermore, the equilibrium constant for cupferron was determined to be 0.076±0.013 mmol/L.