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Structure and Enzymatic Activity of an Intramolecular Complex of Enzyme with Polyelectrolyte

酶与聚电解质分子内复合物的结构和酶活性

基本信息

批准号：
08455434
负责人：
KOKUFUTA Etsuo
金额：
$ 4.29万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

Complexation of proteins with polyelectrolytes is interesting from two points of view. The first concerns the way in which the polymers interact with non-flexible protein molecules. The second concerns the extent to which biochemical activity is maintained in the resulting complexes, the answer to which is central to the molecular design of composite protein-polymer systems, such as immobilized enzymes, as well as to the design of protein separation processes using water-soluble polymers.By combinations of quasi-elastic light scattering (QELS), static light scattering (SLS), electrophoretic light scattering (ELS) techniques, we suggested that polyion and protein give forth an "intrapolymer" complex at a low ionic strength. However, such complexes readily aggregate ; therefore, many difficulties arise in the characterization of intrapolymer complex.Complexes of proteins with neutral polymers are less prone to exhibit such aggregation effects ; thus, the complexation between human serum albumin (HSA) and poly(ethylene glycol) (PEG) was studied. QELS study for aqueous HSA-PEG mixtures at different levels of pH and ionic strength (NaCI) showed the formation of a water-soluble complex, the size of which varied depending on both ionic strength and molecular weight of PEG but remained unaltered when the mixing ratio of PEG to HSA was varied. The study of the complexation in the presence and absence of 1M urea as a function of pH by QELS and fluorescence spectroscopy showed that hydrogen bonding plays an important role in the complex formation. A combination of SLS and dialysis method at pH 2 and at ionic-strength of 0.1 demonstrated that the complexation yields an intrapolymer complex in which several HSA molecules bound to a PEG chain. In addition, ELS indicated that the resulting intrapolymer complex behaves like a free draining coil during electrophoresis.

从两个角度来看，蛋白质与聚电解质的络合是有趣的。第一个问题是聚合物与非柔性蛋白质分子相互作用的方式。第二个问题涉及到在产生的复合物中维持生化活性的程度，这个问题的答案对于复合蛋白质-聚合物系统的分子设计（如固定化酶）以及使用水溶性聚合物的蛋白质分离过程的设计至关重要。结合准弹性光散射（QELS）、静态光散射（SLS）和电泳光散射（ELS）技术，我们认为多离子和蛋白质在低离子强度下形成“聚合物内”复合物。然而，这种复合物很容易聚集；因此，聚合物内配合物的表征出现了许多困难。中性聚合物的蛋白质复合物不太容易表现出这种聚集效应；因此，研究了人血清白蛋白（HSA）与聚乙二醇（PEG）的络合作用。在不同pH和离子强度（NaCI）水平下，HSA-PEG水溶液混合物的QELS研究表明，水溶液复合物的形成取决于PEG的离子强度和分子量，但随着PEG与HSA的混合比例的变化，复合物的大小保持不变。通过QELS和荧光光谱研究了1M尿素存在和不存在情况下的络合作用与pH的关系，结果表明氢键在络合物的形成中起重要作用。在pH为2，离子强度为0.1的条件下，SLS和透析方法的结合表明，络合产生了一个聚合物内络合物，其中几个HSA分子结合在PEG链上。此外，ELS表明，所得聚合物内配合物在电泳过程中表现得像一个自由排水线圈。