Five recombinant fragments of human serum albumin -: Tools for the characterization of the warfarin binding site
Five recombinant fragments of human serum albumin -: Tools for the characterization of the warfarin binding site
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DOI：
10.1110/ps.9.8.1455
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发表时间：
2000-08-01
期刊：
影响因子：
8
通讯作者：
R端ker, F
中科院分区：
文献类型：
--
作者：
Dockal, M;Chang, M;R端ker, F
人血清白蛋白 (HSA) 与大量化学上多样化的配体在特定结合位点相互作用。为了查明在人血清白蛋白上形成华法林结合位点的必要结构元件，制备了一组确定的五种重组蛋白，包含 N 端部分的结构域和/或子结构域的组合，并通过生化标准程序、色氨酸荧光和圆二色性测量进行表征，表明保存完好的二级和三级结构。通过荧光滴定实验估计与华法林结合的亲和常数，发现 HSA-DOM I-II 和 HSA 最高，其次是 HSA-DOM IB-II、HSA-DOM II 和 HSA-DOM I-IIA。此外，还进行了紫外差光谱和诱导圆二色性实验，以深入了解华法林与独立蛋白质片段的结合机制。这项系统研究表明，华法林的主要结合位点集中在子结构域 IIA 中，其中子结构域 IIB 和结构域 I 具有不可或缺的结构贡献，而结构域 III 不参与该结合位点，强调了使用重组片段组合进行研究和准确定位 HSA 上配体结合位点的巨大潜力。
Human serum albumin (HSA) interacts with a vast array of chemically diverse ligands at specific binding sites. To pinpoint the essential structural elements for the formation of the warfarin binding site on human serum albumin, a defined set of five recombinant proteins comprising combinations of domains and/or subdomains of the N-terminal part were prepared and characterized by biochemical standard procedures, tryptophanyl fluorescence, and circular dichroic measurements, indicating well-preserved secondary and tertiary structures. Affinity constants for binding to warfarin were estimated by fluorescence titration experiments and found to be highest for HSA-DOM I-II and HSA, followed by HSA-DOM IB-II, HSA-DOM II, and HSA-DOM I-IIA. In addition, ultraviolet difference spectroscopy and induced circular dichroism experiments were carried out to get an in depth understanding of the binding mechanism of warfarin to the fragments as stand-alone proteins. This systematic study indicates that the primary warfarin binding site is centered in subdomain IIA with indispensable structural contributions of subdomain IIB and domain I, while domain III is not involved in this binding site, underlining the great potential that lies in the use of combinations of recombinant fragments for the study and accurate localization of ligand binding sites on HSA.