Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states
Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states
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DOI：
10.1006/abio.2001.5411
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发表时间：
2001-12-15
影响因子：
2.9
通讯作者：
Philo, JS
中科院分区：
文献类型：
--
作者：
Kendrick, BS;Kerwin, BA;Philo, JS
表征蛋白质-聚合物缀合物的溶液结构和蛋白质-配体相互作用在生物技术和生物化学等领域中是重要的。在线经典光散射（LS），折射率（RI）和紫外检测的尺寸排阻高效液相色谱法提供了一个强大的工具，在这样的表征。提出了利用LS、RI和UV信号快速测定蛋白质缀合物的缀合程度和分子量的新方法。不需要色谱峰的基线分辨率;仅需要充分分离峰以代表相对纯的馏分。还描述了用于测定蛋白质缀合物的仅多肽质量的改进技术。这些技术被应用于确定促红细胞生成素糖基化的程度，聚乙二醇共轭核糖核酸酶A和脑源性神经营养因子的程度，以及这些分子的溶液缔合状态。RI，UV和LS检测器的校准方法也将得到解决，以及在线方法来确定蛋白质消光系数和dn/dc值的非共轭和共轭蛋白质分子。(C)2001年爱思唯尔科学
Characterizing the solution structure of protein-polymer conjugates and protein-ligand interactions is important in fields such as biotechnology and biochemistry. Size-exclusion high-performance liquid chromatography with online classical light scattering (LS), refractive index (RI), and UV detection offers a powerful tool in such characterization. Novel methods are presented utilizing LS, RI, and UV signals to rapidly determine the degree of conjugation and the molecular mass of the protein conjugate. Baseline resolution of the chromatographic peaks is not required; peaks need only be sufficiently separated to represent relatively pure fractions. An improved technique for determining the polypeptide-only mass of protein conjugates is also described. These techniques are applied to determining the degree of erythropoietin glycosylation, the degree of polyethylene glycol conjugation to RNase A and brain-derived neurotrophic factor, and the solution association states of these molecules. Calibration methods for the RI, UV, and LS detectors will also be addressed, as well as online methods to determine protein extinction coefficients and dn/dc values both unconjugated and conjugated protein molecules. (C) 2001 Elsevier Science