Laboratory for Molecular Surfaces and Nanotechnology, Dipartimento di Scienze Chimiche, Università di Catania and CSGI, Viale A. Doria 6, 95125 Catania, Italy.
Langmuir. 2011 Jan 4;27(1):312-9. doi: 10.1021/la104127q. Epub 2010 Dec 9.
The dynamic adsorption of human serum albumin (HSA) and plasma fibronectin (Fn) onto hydrophobic poly(hydroxymethylsiloxane) (PHMS) and the structures of adsorbed protein layers from single and binary protein solutions were studied. Spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation monitoring (QCM-D) together with atomic force microscopy (AFM) were used to measure the effective mass, thickness, viscoelastic properties, and morphology of the adsorbed protein films. Adsorbed HSA formed a rigid, tightly bound monolayer of deformed protein, and Fn adsorption yielded a thick, very viscoelastic layer that was firmly bound to the substrate. The mixed protein layers obtained from the coadsorption of binary equimolecular HSA-Fn solutions were found to be almost exclusively dominated by Fn molecules. Further sequential adsorption experiments showed little evidence of HSA adsorbed onto the predeposited Fn layer (denoted as Fn ≫ HSA), and Fn was not adsorbed onto predeposited HSA (HSA ≫ Fn). The conformational arrangement of the adsorbed Fn was analyzed in terms of the relative availability of two Fn domains. In particular, (4)F(1)·(5)F(1) binding domains in the Hep I fragment, close to the amino terminal of Fn, were targeted using a polyclonal antifibronectin antibody (anti-Fn), and the RGD sequence in the 10th segment, in the central region of the molecule, was tested by cell culture experiments. The results suggested that coadsorption with HSA induced the Fn switch from an open conformation, with the amino terminal subunit oriented toward the solution, to a close conformation, with the Fn central region oriented toward the solution.
研究了人血清白蛋白（HSA）和血浆纤维连接蛋白（Fn）在疏水性聚（羟甲基硅氧烷）（PHMS）上的动态吸附，以及从单一组分和双组分蛋白质溶液中吸附的蛋白质层的结构。光谱椭圆术（SE）和带有耗散监测的石英晶体微天平（QCM-D）以及原子力显微镜（AFM）用于测量吸附蛋白质膜的有效质量、厚度、粘弹性和形态。吸附的 HSA 形成刚性的、紧密结合的变形蛋白质单层，而 Fn 的吸附则产生了厚的、非常粘弹性的层，该层牢固地结合在基底上。从等分子 HSA-Fn 二元共吸附溶液中获得的混合蛋白质层几乎完全由 Fn 分子主导。进一步的顺序吸附实验表明，很少有 HSA 吸附到预沉积的 Fn 层上（表示为 Fn ≫ HSA），并且 Fn 没有吸附到预沉积的 HSA 上（HSA ≫ Fn）。通过相对可用性分析了吸附 Fn 的构象排列两个 Fn 结构域。特别是，接近 Fn 氨基末端的 Hep I 片段中的（4）F（1）·（5）F（1）结合结构域使用多克隆抗纤维连接蛋白抗体（抗-Fn）进行靶向，而分子中心区域的第 10 个片段中的 RGD 序列通过细胞培养实验进行测试。结果表明，与 HSA 的共吸附诱导 Fn 从开放构象转变为闭合构象，氨基末端亚基朝向溶液，而 Fn 中心区域朝向溶液。
