SPR竞争检测PFOS与血清蛋白相互作用的研究-中国化学会.docVIP

SPR竞争检测PFOS与血清蛋白相互作用的研究 高 宇，郭良宏* 中国科学院生态环境研究中心，环境化学与生态毒理学国家重点实验室，100085，北京 E-mail: lhguo@rcees.ac.cn 环境污染物通过与生物大分子的相互作用，可以影响或者改变生物大分子的空间结构，进而影响其功能。研究二者亲和力的强弱以及作用位点对于污染物致毒机理的探索具有重要的意义。 全氟辛烷磺酰基化合物（PFOS）被广泛应用于工业以及生活用品。PFOS与人血清蛋白（HSA）的结合很可能会影响血清蛋白对脂肪酸的转运和代谢。传统的方法如质谱和核磁共振都需要复杂的样品前处理过程，不具备位点特异性。荧光光谱法中结合方式特异的荧光指示剂数目有限，无法得到所有位点的结合信息，对于无法到得IC50的竞争计算不出结合常数。 本研究应用表面等离子体激元共振(surface plasmon resonance, SPR)有快速，免标记，能够实时检测分子结合，高质量的动力学信息的优点。而竞争检测法解决了PFOS难以固定的问题。对于每个位点的检测可以通过封闭其它结合位点来实现。我们首先在金基底模拟了脂肪酸的结构，实现了对HSA的特异性结合，降低了非特异性吸附，并且采用不同浓度的PFOS竞争，得到了PFOS的竞争曲线。通过数学公式的推导和计算，得到了表面竞争反应中PFOS与HSA的结合常数,其与脂肪酸的亲和力相当。CD光谱的实验结果证实了PFOS与HSA的结合会导致蛋白的二级结构发生明显的改变。该方法为实现PFOS与HSA各位点结合动力学常数的测量奠定了基础。 [1] Yan-Min Chen, Liang-Hong Guo, Archives of Toxicology, 2009, 83, 255-261 [2] Xing Han, Timothy A. snow, Gary W. Jepson, Chem.Res.Toxicol., 2003, 16, 775-781 Studies on the Binding Interaction between PFOS and HSA by Using SPR Competitive Assay Yu Gao, Liang-Hong Guo* State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, 100085, Beijing The binding interaction between pollutants and bio-macromolecule is a key step in its toxic process. Perfluorooctane sulfonate (PFOS) has been used in industrial and consumer products. However, because of the similar molecular structure of PFOS and fatty acid, the binding interaction between PFOS and HSA is likely to affect the transport and metabolism of fatty acid. We proposed here a competitive assay strategy based on surface plasmon resonance spectroscopy. Fatty acid was immobilized on the gold substrate to achieve specific binding of HSA. To calculate the competitive curves of PFOS, the binding constant of PFOS and HSA was finally obtained. PFOS was found to have the similar affinity and binding sites as fatty acids. The results of CD spectrum reveal that PFOS can obviously change the secondary structure of HSA.