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目的建立磷脂化重组人铜锌超氧化物歧化酶(PC-SOD)中蛋白和胆碱含量的测定方法,并计算PC-SOD的修饰率。方法采用凯氏定氮法,即通过测定供试品的总氮含量及经钨酸沉淀去除蛋白质的供试品滤液中的非蛋白氮含量,计算PC-SOD蛋白氮含量,并验证方法的准确性和重复性;应用磷脂C检测试剂盒,经碱水解显色法检测PCSOD的胆碱含量,并验证方法的准确性和重复性。通过PC-SOD中的蛋白氮和胆碱含量计算其修饰率。同时采用该方法对3批PC-SOD供试样品进行检测。结果凯氏定氮法的回收率为95%~105%,RSD为0.59%;碱水解显色法的回收率为95%~105%,RSD为1.65%。3批PC-SOD供试样品的修饰率分别为4.46、4.33和4.28。结论该方法操作简便,具有良好的准确性及重复性,可用于PC-SOD修饰率的检测。
Objective To establish a method for the determination of protein and choline in phospholipid-modified recombinant human copper-zinc-superoxide dismutase (PC-SOD) and calculate the rate of PC-SOD modification. Methods The Kjeldahl method was used to calculate the nitrogen content of PC-SOD protein by measuring the total nitrogen content of the test sample and the non-protein nitrogen content in the filtrate of the test product by removing the protein by tungstic acid precipitation. The method was accurate Sex and repeatability. The phospholipid C detection kit was used to detect the choline content of PCSOD by alkaline hydrolysis and colorimetry, and the accuracy and repeatability of the method were verified. The modification rate was calculated by the protein nitrogen and choline content in PC-SOD. At the same time, three batches of PC-SOD test samples were tested by this method. Results The recovery rate of Kjeldahl method was 95% ~ 105%, RSD was 0.59%. The recovery of alkali hydrolysis was 95% ~ 105% with RSD of 1.65%. The modification rates of three batches of PC-SOD samples were 4.46, 4.33 and 4.28, respectively. Conclusion The method is simple and convenient, with good accuracy and repeatability, which can be used to detect the rate of PC-SOD modification.