超氧化物歧化酶的固定及其活性检测

目的探讨超氧化物歧化酶(SOD)的固定及固定化SOD活性的检测方法。方法以淀粉为载体,对SOD进行固定,采用改进的邻苯三酚测活法(以Vc为终止剂),并结合过滤或离心的方法去除沉淀,测定固定化SOD的活性。比较过滤法与离心法,及过滤法与经典邻苯三酚法测定酶活性的差异,并检测过滤法的线性、精密性和准确性。结果过滤法检测SOD活性的精密性较离心法高,与经典邻苯三酚法检测结果差异无统计学意义,在SOD浓度为6~60μg/ml范围内,SOD活性值与浓度线性关系良好(r=0.999 5),精密性及准确性良好。固定化SOD的比活性为1 730.07 U/g,酶活回收率为53.3%。结论以淀粉为载体固定化SOD效果好,经改进的邻苯三酚法可用于固定化SOD的活性测定。

Immobilization and Activity of Superoxide Dismutase

Objective To investigate the immobilization of superoxide dismutase(SOD)and develop a method for determina-tion the activity of immobilized SOD.Methods SOD was immobilized using starch as a carrier and determined by an improved pyro-gallol autoxidation method using Vc as a terminator,in which the precipitate was removed by filtration or centrifugation.The determi-nation result by the improved method including filtration step was compared with those by improved method including centrifugation step and by classic pyrograllol autoxidation method,based on which the linearity,precision and accuracy of the improved method was evaluated.Results The improved pyrogallol autoxidation method including filtration step showed high precision as compared with that including centrifugation step.The determination result by the method showed no significant difference with that by classic pyro-gallol autoxidation method.The SOD activity showed good linear relationship to SOD concentration at a range of 6 ~ 60 μg / ml(r = 0.999 5).The improved method also showed good precision and accuracy.The specific activity and recovery of immobilized SOD were 1 730.07 U / g and 53.3% respectively.Conclusion Starch is suitable for the immobilization of SOD,and the improved pyro-gallol autoxidation method including filtration step may be used for determination of activity of immobilized SOD.