Microenvironmental Analysis of Drug Binding Sites on Human Serum Albumin and alpha_1-Acid Glycoprotein

本研究旨在利用不同的荧光探针，明确人血清白蛋白（HSA）和α _1-酸性糖蛋白（AGP）药物结合位点的特点，这些荧光探针的结合位点分别针对HSA和AGP。所得结果如下：(1)疏水作用力是药物与HSA和AGP结合的驱动力，从结合亲合力与配分系数的相关性、热力学分析和光谱分析均可看出。此外，唾液酸被发现在药物与AGP的结合中起一定作用。(2)从荧光探针位移数据来看，HSA上有3个药物结合位点，AGP上有1个加宽的柔性药物结合位点。虽然证实了HSA上存在三个不同的结合位点，但我们有理由认为这些位点并不是完全分离的，而是明显重叠并相互影响的。(3) HSA上药物结合位点的疏水性顺序为：II位点>位点I>位点eiii。对于AGP，证实了一个药物结合区，但酸性药物结合位点的疏水性大于碱性药物结合位点。此外，发现HSA和AGP上的位点的结合大小不同。此外，新开发的微粘度分析方法也表明，在HSA的所有药物结合位点中，I位点的柔韧性最小，酸性药物结合位点比碱性药物结合位点更具柔韧性。

The present study was undertaken in a view to clarify the characteristics of the drug binding sites on human serum albumin(HSA)and alpha_1-acid glycoprotein(AGP)using different fluorescent probes whose binding are site specific to HSA and AGP. Results obtained are as follows.(1)Hydrophobic forces were the driving forces for the binding of drugs to both HSA and AGP as evidenced from the obtained correlation of binding affinities with partition coefficients, the thermodynamic analysis and the spectral analysis. Further, sialic acid was found to impart some role in the binding of drugs to AGP.(2)From the displacement data using the fluorescent probes it seems that there are three drug binding sites on HSA andone widened flexible drug binding site on AGP, respectively. Although the presence of three different binding sites on HSA is evidenced, it is rather reasonable to consider that these sites are not completely separated but significantly overlapped and influenced by each other.(3)The hydrophobicity of the drug binding sites on HSA were in the order of site II>site I>siteIII. For AGP, one drug binding area was evidenced, however, the hydrophobicity of the acidic drug binding site was found to be greater than that of basic drug binding site. Moreover, the binding sizes of the sites on HSA or AGP were found to be different. In addition, the microviscosity analysis, a newly developed method, also suggested that the flexibility of the site I was the smallest among all the drug binding sites on HSA and the acidic drug binding site was more flexible as compared with the basic drug binding site.