鲎血蓝蛋白的分离纯化及其酚氧化酶活性的初步研究

Separation and Purification of Hemocyanin from Limulus and Its Phenoloxidase Activity

采用饱和硫酸铵盐析法、透析除盐和丙烯葡聚糖凝胶柱层析分离纯化鲎血蓝蛋白,通过紫外可见吸收光谱和SDS-PAGE图谱对其进行定性分析,再通过单因素实验和响应面实验研究了温度、pH值、邻苯二酚与鲎血蓝蛋白浓度比、时间等因素对鲎血蓝蛋白酚氧化酶活性的影响。结果表明,纯化后的鲎血蓝蛋白在280 nm、340 nm处有特征吸收峰,有2个蛋白亚基,分子量约为66 kDa,为典型的节肢动物血蓝蛋白;在温度为36.7℃、pH值为6.8、邻苯二酚与鲎血蓝蛋白浓度比为1.7∶1、时间为2.1 min的最佳条件下,鲎血蓝蛋白酚氧化酶活性最高,为83.3 U·min^(-1)·mL^(-1)。

We separated and purified the hemocyanin from Limulus by saturated ammonium sulfate salting-out method,dialysis desalting method,and allyl dextran gel column chromatograph;meanwhile,we qualitatively analyzed the purified hemocyanin by UV-Vis absorption spectrum and SDS-PAGE profile.Moreover,we studied the effects of temperature,pH value,concentration ratio of 1,2-benzenediol to hemocyanin from Limulus,and time on the phenoloxidase activity of hemocyanin from Limulus by single-factor experiments and response surface methodologies.The results show that the purified hemocyanin from Limulus have characteristic absorption peaks at 280 nm and 340 nm,and have two protein subunits with the molecular weight of about 66 kDa,which is a typical arthropod hemocyanin.The purified hemocyanin shows the highest phenoloxidase activity of 83.3 U·min^(-1)·mL^(-1) under the conditions as follows:the temperature is 36.7℃,the pH value is 6.8,the concentration ratio of 1,2-benzenediol to hemocyanin from Limulus is 1.7∶1,and the time is 2.1 min.