Molecular Characterization of Multivalent Bioconjugates by Size-Exclusion Chromatography with Multiangle Laser Light Scattering
Molecular Characterization of Multivalent Bioconjugates by Size-Exclusion Chromatography with Multiangle Laser Light Scattering
复制标题
DOI：
10.1021/bc3000595
复制
发表时间：
2012-09-01
影响因子：
4.7
通讯作者：
Healy, Kevin E.
中科院分区：
文献类型：
--
作者：
Pollock, Jacob F.;Ashton, Randolph S.;Healy, Kevin E.
生物缀合物反应产物的取代度和化合价通常判断不佳或需要多种耗时和产物消耗的化学表征方法。当分析和优化昂贵的多价生物活性缀合物的效力时，这些方面变得至关重要。在这项研究中，将具有多角度激光散射的尺寸排阻色谱与折射率检测和紫外光谱（SEC-MALS-RI-UV）配对，以表征肽或蛋白质与生物聚合物支架缀合的产物的反应效率、取代度和化合价，即，透明质酸（HyA）。与蛋白质定量测定的估计相比，分子表征更完整，并且该方法的利用导致聚合物生物缀合物的分子结构的更准确的推导。使用该技术获得的信息可以改进大分子工程设计原理，并有助于更好地理解生物系统中的多价大分子相互作用。
The degree of substitution and valency of bioconjugate reaction products are often poorly judged or require multiple time- and product-consuming chemical characterization methods. These aspects become critical when analyzing and optimizing the potency of costly polyvalent bioactive conjugates. In this study, size-exclusion chromatography with multiangle laser light scattering was paired with refractive index detection and ultraviolet spectroscopy (SEC-MALS-RI-UV) to characterize the reaction efficiency, degree of substitution, and valency of the products of conjugation of either peptides or proteins to a biopolymer scaffold, i.e., hyaluronic acid (HyA). Molecular characterization was more complete compared to estimates from a protein quantification assay, and exploitation of this method led to more accurate deduction of the molecular structures of polymer bioconjugates. Information obtained using this technique can improve macromolecular engineering design principles and help to better understand multivalent macromolecular interactions in biological systems.