生物分离法制备细胞色素C质量的影响因素

Factors affecting the quality of cytochrome C prepared by biological separation

细胞色素C为临床上应用广泛的生化药品。为了提高细胞色素C的质量,对制备过程中影响细胞色素C质量的因素进行考察,采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳法(SDS PAGE)对不同工艺制备的细胞色素C进行纯度分析,并对SDS PAGE图谱中约2.5×10^4的蛋白质进行液相色谱串联质谱法(LC MS/MS)分析与鉴定。结果表明:在SDS PAGE电泳图中,使用三氯乙酸(TCA)沉淀剂的传统工艺制备的细胞色素C样品除了有1.22×10^4的细胞色素C,还包括1个约2.5×10^4的蛋白分子;而不使用三氯乙酸沉淀剂的新工艺制备的细胞色素C样品,仅有1.22×10^4的细胞色素C,而未发现约2.5×10^4的蛋白。SDS PAGE图谱中分子量2.5×10^4的蛋白分子的LC MS/MS分析与鉴定结果表明其与猪源细胞色素C覆盖率最大(达到76%),结合其分子量大小,可以证明该蛋白为细胞色素C二聚体。细胞色素C制备过程中三氯乙酸沉淀剂易导致细胞色素C形成二聚体,故三氯乙酸是影响细胞色素C质量的关键因素之一。

Cytochrome C is a biochemical drug widely used in clinical treatment.To improve the quality of cytochrome C,we studied the factors influencing the quality of cytochrome C in the preparation process.The purity of cytochrome C prepared by different processes was analyzed by SDS-PAGE,and the 2.5×104 protein in SDS-PAGE was analyzed and identified by LC-MS/MS.The results of SDS-PAGE electrophoresis show that the sample of cytochrome C prepared by traditional process using trichloroacetic acid(TCA)precipitator contained not only 1.22×10^4 cytochrome C,but also a protein molecule of about 2.5×10^4.Cytochrome C samples prepared by the new process without trichloroacetic acid precipitator only had 1.22×10^4 cytochrome C,but no 2.5×10^4 protein was found.The LC-MS/MS analysis and identification result of the 2.5×10^4 protein in the SDS-PAGE map show that the coverage between this protein and cytochrome C(susscrofa)was 76%.Combining with its molecular weight,it can be proved that this protein is cytochrome C dimer.TCA precipitator during the preparation of cytochrome C easily leads to the formation of cytochrome C dimer.Therefore,TCA is one of the key factors affecting the quality of cytochrome C.