The location of the high- and low-affinity bilirubin-binding sites on serum albumin: Ligand-competition analysis investigated by circular dichroism
The location of the high- and low-affinity bilirubin-binding sites on serum albumin: Ligand-competition analysis investigated by circular dichroism
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DOI：
10.1016/j.bpc.2013.06.004
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发表时间：
2013-10-01
影响因子：
3.8
通讯作者：
Urbanova, Marie
中科院分区：
文献类型：
--
作者：
Goncharova, Iryna;Orlov, Sergey;Urbanova, Marie
在人（HSA）、牛（BSA）、大鼠（RSA）、兔（RbSA）和绵羊（SSA）五种哺乳动物血清白蛋白（SA）中，三个具有不同亲和力的胆红素（BR）结合位点分别位于IB、HA和IIIA亚结构域。在BR/SA = 1/1体系中，用圆二色性光谱（CD）分析了高亲和性BR结合位点的立体选择性，用差示CD分析了低亲和性位点。布洛芬（亚结构域MA的标记物）和氯高铁血红素（亚结构域IB的标记物）的位点特异性配体竞争实验未显示BR/SA = 1/1系统的任何变化，并显示在BR/SA = 3/1时结合BR减少。两个位点均被鉴定为对BR具有低亲和力的位点。Arg-Lys残基的立体选择性和安排之间的相关性表明BR-结合位点之间的相似性在子域IIIA的所有SA研究。亚结构域IB在HSA、BSA、SSA和RbSA中对BR具有P-立体选择性，而在RSA中对BR具有M-立体选择性。与棉酚的配体竞争实验表明，BR/SA = 1/1系统以及BR/SA = 3/1系统的结合BR的CD信号降低。HA亚结构域被指定为高亲和力BR结合位点。HSA（和RSA，RbSA）中该位点的P-立体选择性是由带电残基R257/R218-R222的右侧定位引起的，而R257/R218-R199的左侧定位导致BSA（和SSA）中主要结合位点的M-立体选择性。(C)2013爱思唯尔有限公司版权所有。
The locations of three bilirubin (BR)-binding sites with different affinities were identified as subdomains IB, HA and IIIA for five mammalian serum albumins (SAs): human (HSA), bovine (BSA), rat, (RSA), rabbit (RbSA) and sheep (SSA). The stereoselectivity of a high-affinity BR-binding site was identified in the BR/SA = 1/1 system by circular dichroism (CD) spectroscopy, the sites with low affinity to BR were analyzed using difference CD. Site-specific ligand-competition experiments with ibuprofen (marker for subdomain MA) and hemin (marker for subdomain IB) did not reveal any changes for the BR/SA = 1/1 system and showed a decrease of the bound BR at BR/SA = 3/1. Both sites were identified as sites with low affinity to BR. The correlation between stereoselectivity and the arrangement of Arg-Lys residues indicated similarity between the BR-binding sites in subdomain IIIA for all of the SAs studied. Subdomain IB in HSA, BSA, SSA and RbSA has P-stereoselectivity while in RSA it has M-selectivity toward BR. A ligand-competition experiment with gossypol shows a decrease of the CD signal of bound BR for the BR/SA = 1/1 system as well as for BR/SA = 3/1. Subdomain HA was assigned as a high-affinity BR-binding site. The P-stereoselectivity of this site in HSA (and RSA, RbSA) was caused by the right-hand localization of charged residues R257/R218-R222, whereas the left-hand orientation of R257/R218-R199 led to the M-stereoselectivity of the primary binding site in BSA (and SSA). (C) 2013 Elsevier B.V. All rights reserved.