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很有趣。第一个涉及聚合物与非氟化蛋白分子相互作用的方式。第二个问题是关注生物化学活性在产生的复合物中的多大程度，其答案对于复合蛋白质聚合物系统的分子设计至关重（ELS）技术，我们建议在低离子强度下，Polyion和蛋白质给出了“内聚合物”复合物。但是，这样的复合物很容易聚集。因此，在内外聚合物复合物的表征中出现了许多困难。因此，研究了人血清白蛋白（HSA）和聚乙二醇（PEG）之间的络合。 QELS研究在不同水平的pH和离子强度（NACI）的HSA-PEG水溶液中表明了水溶性络合物的形成，其大小取决于PEG的离子强度和分子量，但在PEG与HSA混合比时保持不变，但保持不变。在存在和不存在1M尿素作为Qel的pH和荧光光谱的情况下，对络合的研究表明，氢键在复合形成中起重要作用。在pH 2和0.1的离子强度下，SLS和透析方法的组合表明，络合物产生了一个内聚合物复合物，其中几种HSA分子与PEG链结合。此外，EL表明所产生的内聚合物复合物的表现就像电泳过程中的自由排水线圈。