光裸方格星虫纤溶活性蛋白的提取及抗氧化活性研究

Extraction and antioxidant activity of the protein fraction with fibrinolytic activity from Sipunculus nudus Linnaeus

目的:从光裸方格星虫提取纤溶活性蛋白并研究其抗氧化能力。方法:光裸方格星虫全虫匀浆后,用3倍体积PBS缓冲液(0.02 mol/L,p H7.5)在4℃条件下抽提6 h,上清液用硫酸铵盐析法粗提纯纤溶活性蛋白;采用普鲁士蓝法测定该蛋白组分的还原能力,在DPPH体系,Fenton体系和邻苯三酚自氧化体系中分别测定对DPPH自由基(DPPH·)、羟基自由基(·OH)、超氧阴离子自由基(O_2^-·)的清除能力。结果:用30%饱和度硫酸铵沉淀杂质,再用50%饱和度硫酸铵沉淀上清液的纤溶活性蛋白可取得较好的纯化效果,该蛋白组分具有一定还原能力,具有显著的清除DPPH·和·OH作用,且与剂量呈正相关,EC_(50)分别为1.23、6.07 mg/m L,对O_2^-·则有负清除作用。结论:采用两级硫酸铵盐析纯化的光裸方格星虫纤溶活性蛋白具有一定体外抗氧化活性。

Objective：To extract the protein fraction with fibrinolytic activity from Sipunculus nudus Linnaeus and evaluate the antioxidant activity.Methods-Fresh Sipunculus nudus were precipitated with PBS buffer（0.02 mol/L, pH7.5） to homogenize and the clear supernatant of tissue homogenate was salting out with stepwise ammonium sulfate to obtain the protein fraction with highly fibrinolytic enzyme activity.The antioxidant activity was investigated by using methods of purssian blue reaction, DPPH, phenanthrorine and pyrogallol autoxidation.Result：The protein fraction with highly fibrinolytic enzyme activity was obtained by using 30% saturation ammonium sulfate to remove the mixed protein, and then by 50% saturated ammonium sulfate to obtain the target protein precipitation. The protein fraction had reduction ability and strong scavenging effect on DPPH· and · OH.The concentration of scavenging 50% （EC50）of DPPH· and· OH were 1.23 mg/mL and 6.07 mg/mL respectively. Whereas it had significant negative scavenging ability on O2- ·.Conclusion： The protein fraction with fibrinolytic activity from Sipunculus nudus Linnaeus obtained by two steps ammonium sulfate salting out method had certain antioxidant capacity.