一步法和两步法毛细管等电聚焦测定蛋白质和多肽等电点的比较

利用一步法和两步法毛细管等电聚焦(cIEF)方法分离测定了蛋白质和多肽的等电点(pI)。讨论了两步法等电聚焦过程所需的溶液组成、样品进样体积、聚焦电压、聚焦时间和分离条件等因素对分离效果的影响。并对一步法和两步法进行了比较。对细胞色素C、血红蛋白、肌红蛋白、转铁蛋白和牛血清白蛋白以及6种多肽的分析结果表明:一步法步骤简单,分离速度快,可测定单一组分的pI,也能快速分离混合蛋白和多肽,但分离度较差,且不能同时准确测定各组分的pI;两步法步骤复杂,分析时间较长,但能够同时分离并准确测定混合样品中各组分的pI,所测的pI值与单一组分进行测定的结果基本一致。两种方法可相互结合、互为补充,可广泛应用于两性生物微粒等电点的快速和准确测定。

Comparison of one-step and two-step methods for pI determination of proteins and polypeptides by capillary isoelectric focusing

One-step and two-step capillary isoelectric focusing (cIEF) methods were employed for the separation and pI determination of proteins and polypeptides. The parameters affecting the analysis efficiency, such as the sample solution, injection volume, focusing voltage, focusing time and driving conditions were optimized. The comparison of the two methods for separation of cytochrome C, hemoglobin, myoglobin, transferrin, bovine serum albumin and six polypeptides showed that the one-step cIEF was simple and fast, which could determine the pI of single component as well as it was rapid for protein and polypeptide separation, but it could not get good resolution or accurate pI of each component in a mixed sample. The two-step cIEF was more complex and needed longer time, however, which could separate and exactly determine the pI of each component in the mixture, and the pI value of each component determined was consistent with that determined using a single sample. The two methods are complementary, and can be widely used in rapid and accurate determination of the pI of amphiphilic biological particles.