Wilchek Meir, Miron Talia
Department of Biological Chemistry, Wiezmann Institue of Science, Rehovot 76100, Israel.
Bioconjug Chem. 2015 Mar 18;26(3):502-10. doi: 10.1021/bc5006152. Epub 2015 Feb 25.
Our objective was to develop a method mimicking the natural process of coherence in marine mollusks, by direct chemical conversion of protein tyrosine residues to DOPA-o-quinones, which consequently generates polymerization and cross-linking. Fremy's salt, (ON(SO3K)2, was used to convert tyrosine residues in peptides and proteins to reactive o-quinones. The conversion of tyrosines to DOPA-o-quinones, and their ability to polymerize or cross-link, was tested on tyramine, peptides, and proteins. The peptides tested were as follows: biotin-PEG4-tyramine (PEG-BT), and two decapeptides (identical to the repeating units comprising the mussel's adhesive protein). The proteins tested were as follows: bovine pancreatic ribonuclease A (RNase), lysozyme, IgG, avidin, and streptavidin. The oxidized peptides and proteins were all shown to incorporate oxygen atoms and undergo polymerization and cross-linking, depending on the availability of nucleophiles, mostly lysine amino groups of proteins. All the peptides and the noninteracting proteins such as RNase and lysozyme underwent homopolymerization upon Fremy's salt oxidation. When Fremy's salt oxidaized PEG-BT was mixed with the above proteins, it did not react with any of these proteins because PEG-BT underwent fast self-polymerization. Conversely, streptavidin or avidin cross-linked with PEG-BT after preincubation, thus showing that biorecognition is a prerequisite for cross-linking. Polymerization and cross-linking also occurred, following Fremy's salt oxidation of interacting proteins such as avidin and strepavidin with biotinyilated lysozyme or biotinylated RNase. This indicates that only proteins in very close proximity readily cross-link and polymerize via tyrosine residues. Attempts to convert DOPA-quinone to DOPA by reduction with sodium dithionite (Na2S2O4), was successful as far as small peptides were used. Fremy's salt oxidation can serve as an easy and useful tool to polymerize and cross-link proteins, for fabrication of biomaterials and to study protein-protein interactions.
我们的目标是开发一种模拟海洋软体动物中自然凝聚过程的方法，即通过将蛋白质酪氨酸残基直接化学转化为多巴 - o - 醌，从而引发聚合和交联反应。使用弗勒密盐（ON(SO3K)2）将肽和蛋白质中的酪氨酸残基转化为活性邻醌。在酪胺、肽和蛋白质上测试了酪氨酸向多巴 - o - 醌的转化及其聚合或交联能力。所测试的肽如下：生物素 - PEG4 - 酪胺（PEG - BT）以及两种十肽（与构成贻贝粘附蛋白的重复单元相同）。所测试的蛋白质如下：牛胰核糖核酸酶A（RNase）、溶菌酶、免疫球蛋白G（IgG）、抗生物素蛋白和链霉抗生物素蛋白。氧化后的肽和蛋白质均显示会结合氧原子并发生聚合和交联反应，这取决于亲核试剂的可用性，主要是蛋白质的赖氨酸氨基。所有的肽以及诸如RNase和溶菌酶等非相互作用蛋白质在弗勒密盐氧化后均发生均聚反应。当弗勒密盐氧化的PEG - BT与上述蛋白质混合时，它不会与这些蛋白质中的任何一种发生反应，因为PEG - BT会快速自聚合。相反，链霉抗生物素蛋白或抗生物素蛋白在预孵育后会与PEG - BT交联，这表明生物识别是交联的先决条件。在抗生物素蛋白和链霉抗生物素蛋白与生物素化的溶菌酶或生物素化的RNase等相互作用蛋白质经弗勒密盐氧化后，也会发生聚合和交联反应。这表明只有非常接近的蛋白质才容易通过酪氨酸残基进行交联和聚合。就小肽而言，尝试用连二亚硫酸钠（Na2S2O4）将多巴醌还原为多巴是成功的。弗勒密盐氧化可作为一种简便且有用的工具，用于蛋白质的聚合和交联，以制造生物材料并研究蛋白质 - 蛋白质相互作用。
