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首页> 外文学位 >Identification of fatty acid and bilirubin binding sites on human serum albumin by 2D-NMR spectroscopy.
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Identification of fatty acid and bilirubin binding sites on human serum albumin by 2D-NMR spectroscopy.

机译：通过2D-NMR光谱鉴定人血清白蛋白上的脂肪酸和胆红素结合位点。

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摘要

In circulation human serum albumin (HSA) is the principal carrier for endogenous lipophilic compounds, primarily non-esterified long chain fatty acids (FA). Since FA bind multiple binding sites with varying affinities, it would be useful to probe the relative affinities for FA binding sites with a method that distinguishes individual binding sites. NMR spectroscopy is a powerful approach for studying interactions of FA with HSA and FA-competitive drugs. In the physiologically relevant solution state, 2D-NMR provides a unique view of HSA-ligand binding in a site-specific manner. Here we show nine, well-resolved peaks in 1H-13C-NMR spectra of 18-13 C-oleic acid (OA)/HSA complexes. Different NMR signals arise, representing FA bound at different sites throughout the protein, with the varying intensities corresponding to the different relative affinities of HSA for OA. This investigation probes the site-specific FA binding sites with four approaches: (i) observation of order of filling with increasing FA molar ratios to HSA; (ii) addition of FA acceptors to observe the dissociation of FA from HSA; (iii) addition of drugs known to bind to low affinity FA sites; and (iv) development and use of HSA mutants that disrupt the binding of FA at high affinity sites. From the order of filling, the three highest affinity-binding sites are clearly differentiated from the six lower/medium affinity-binding sites at the physiologically relevant FA:HSA molar ratio- 4:1. Methyl-beta-cyclodextrin (MbetaCD) extracted FA from individual sites, in a concentration dependent manner, with the highest concentrations removing FA from the highest affinity sites. Relative affinities determined as above were consistent with the binding of drugs to previously defined primary drug binding sites, which displaced bound FA from specific lower affinity sites. HSA mutants were successfully expressed and purified, but FA binding to these proteins did not yield interpretable data. The other aim of this study was to identify the binding location of bilirubin on HSA, for it remains elusive despite intensive study. Unlabeled bilirubin competition suggested binding at primary drug binding sites, while directly probing the bilirubin-binding site with 13C-bilirubin analogs suggested binding in subdomain IB. These studies provide a methodological approach for further analysis of site-specific binding on HSA.
机译：在循环中，人血清白蛋白（HSA）是内源性亲脂性化合物（主要是未酯化的长链脂肪酸（FA））的主要载体。由于FA以不同的亲和力结合多个结合位点，因此用区分单个结合位点的方法探测FA结合位点的相对亲和力将是有用的。 NMR光谱学是研究FA与HSA和FA竞争性药物相互作用的有效方法。在生理相关溶液状态下，2D-NMR以位点特异性方式提供了HSA-配体结合的独特视图。在这里，我们在18-13 C-油酸（OA）/ HSA配合物的1H-13C-NMR光谱中显示了9个分辨良好的峰。出现不同的NMR信号，代表FA结合在整个蛋白质的不同位点，强度的变化对应于HSA对OA的不同相对亲和力。该研究通过四种方法探究了位点特异性FA结合位点：（i）观察FA与HSA摩尔比增加时填充顺序（ii）加入FA受体以观察FA与HSA的分离； （iii）添加已知与低亲和力FA位点结合的药物； （iv）开发和使用破坏高亲和力部位FA结合的HSA突变体。从填充的顺序来看，三个最高的亲和力结合位点与生理学上相关的FA：HSA摩尔比为4：1的六个较低/中等的亲和力结合位点明显不同。甲基-β-环糊精（MbetaCD）以浓度依赖性方式从各个位点提取FA，最高浓度可从最高亲和力位点去除FA。如上确定的相对亲和力与药物与先前定义的初级药物结合位点的结合一致，该结合使结合的FA从特定的较低亲和力位点移位。 HSA突变体已成功表达和纯化，但FA结合这些蛋白质无法产生可解释的数据。这项研究的另一个目的是确定胆红素在HSA上的结合位置，尽管进行了深入研究，但仍然难以捉摸。未标记的胆红素竞争提示在主要药物结合位点结合，而直接用13C-胆红素类似物探测胆红素结合位点则提示在亚结构域IB中结合。这些研究为进一步分析HSA上的位点特异性结合提供了一种方法学方法。

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  • 作者

    Krenzel, Eileen Susan.;

  • 作者单位

    Boston University.;

  • 授予单位 Boston University.;
  • 学科 Biophysics General.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 340 p.
  • 总页数 340
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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