重组碱性成纤维细胞生长因子游离巯基的测定分析

建立了一种紫外-可见分光光度检测法,可以快速、简便、稳定地测定重组碱性成纤维细胞生长因子的游离巯基含量。将检测试剂、标准品及待测样品一起反应后,在412 nm波长处读取OD值,以标准品浓度为横坐标,以吸光值为纵坐标,绘制标准曲线,根据标准曲线计算待测样品中游离巯基含量;同时对该方法进行了方法学验证。结果表明:该方法专属性强,游离巯基含量测定范围为20~80 μg/ml,线性相关系数在0.999以上,回收率范围为91.7%~107.4%;而且该法精密度较好,精密度的变异系数范围为5%~8%。测得的分子表面和总游离巯基含量与理论一致,可用于蛋白质游离巯基检测,以监测分子结构的正确性与均一性。

Measurement and Analysis of Recombinant Basic Fibroblast Growth Factor's Free Sulfhydryl

A rapid and simple analysis method based on uv-vis spectrophotometry technology was established to measure the concentration of free sulfhydryl groups in recombinant basic fibroblast growth factor. Detection reagents,standard references and samples were mixed, incubated for some time,followed by OD₄₁₂ value reading with uv-vis spectrometer. Standard curve was generated by taking the concentration of standard reference as the horizontal coordinate, taking the OD₄₁₂ values as the vertical coordinate. The concentration of free sulfhydryl groups in tested samples was calculated according to standard curve. The method qualification results showed that the method is free sulfhydryl specific, with accuracy range 91.7%~107.4%, precision range 5%~8%, and linearity range 20~80μg/ml. The measured result of free sulfhydryl groups on molecular surface or total were consistent with the theoretical value.This method can be used to measure the free sulfhydryl groups in proteins to monitor the correctness and homogeneity of molecular structure during the quality research of related biopharmaceuticals development.