Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs)
Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs)
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DOI：
10.1039/c4cp01798f
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发表时间：
2014-01-01
影响因子：
3.3
通讯作者：
Reddy, Subrayal M.
中科院分区：
文献类型：
--
作者：
EL-Sharif, Hazim F.;Hawkins, Daniel M.;Reddy, Subrayal M.
利用丙烯酰胺类单体制备了几种蛋白质(血红蛋白、肌红蛋白和过氧化氢酶)的水凝胶分子印迹聚合物。在平衡条件下和一定浓度范围内，利用Hill斜率饱和曲线的特定结合，研究了蛋白质对水分子印迹聚合物的亲和力。我们报道了在微摩尔范围内的平衡解离常数(K-d)(在基于聚丙烯酰胺的MIP中，血红蛋白、肌红蛋白和过氧化氢酶分别为25+/-4µM、44+/-3µM、17+/-2µM)的结合亲和力。对非靶蛋白的非特异性结合或交叉选择性的程度也进行了评估。结果表明，同源蛋白和非同源蛋白对分子印迹聚合物的选择性和亲和力均依赖于其结构和大小的浓度和互补性。这初步归因于在高蛋白质浓度下，在聚合和重结合阶段都形成了蛋白质复合体。我们已经使用原子力光谱来表征MIP腔中的分子相互作用，使用的是蛋白质修饰的AFM尖端。得到了MIP和NIP(非印迹聚合物)表面(在蛋白质负载和卸载状态下)的吸引力和排斥力曲线。我们的作用力数据表明，我们在分子印迹聚合物(MIP)中为模板蛋白产生了选择性空穴，并且我们已经能够量化非特定蛋白质在例如非印迹聚合物(NIP)控制表面上的结合程度。
Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (K-d) within the micro-molar range (25 +/- 4 mu M, 44 +/- 3 mu M, 17 +/- 2 mu M for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface.