Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin.
Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin.
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DOI：
10.1016/j.jchromb.2015.09.041
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发表时间：
2016-05-15
期刊：
影响因子：
--
通讯作者：
Hage DS
中科院分区：
文献类型：
--
作者：
Anguizola J;Debolt E;Suresh D;Hage DS
人血清白蛋白(HSA)的主要内源性配体是非酯化脂肪酸，通常有0.1-2摩尔的脂肪酸与HSA结合。在II型糖尿病中，血清中的脂肪酸水平通常会升高，而高糖的存在会导致HSA的非酶糖基化增加。用高效亲和层析法研究了糖基化和长链脂肪酸对人血清白蛋白与R-华法林和L-色氨酸结合的联合作用(即在该蛋白上药物的主要结合部位--苏德罗位点I和II的探针)。用区带洗脱竞争法研究了肉豆蔻酸、棕榈酸和硬脂酸与这些探针在人血清白蛋白上的相互作用。研究发现，所有这些脂肪酸都与R-华法林在正常HSA和糖化HSA的苏德罗I位有直接竞争，糖化的HSA通常与该位点的脂肪酸有较强的结合。当使用正常人血清白蛋白时，所有脂肪酸与L-色氨酸都有直接竞争，而糖化的人血清白蛋白与L-色氨酸没有竞争或正的变构相互作用。这些数据表明，糖基化可以改变药物和HSA上特定结合部位的脂肪酸的相互作用。这项研究的结果应该有助于更好地理解这些相互作用在糖尿病期间可能发生的变化，并展示如何使用HPAC来检查复杂系统中的药物/溶质-蛋白质相互作用。
The primary endogenous ligands of human serum albumin (HSA) are non-esterified fatty acids, with 0.1–2 moles of fatty acids normally being bound to HSA. In type II diabetes, fatty acid levels in serum are often elevated, and the presence of high glucose results in an increase in the non-enzymatic glycation of HSA. High-performance affinity chromatography (HPAC) was used to examine the combined effects of glycation and the presence of long chain fatty acids on the binding of HSA with R-warfarin and L-tryptophan (i.e., probes for Sudlow sites I and II, the major sites for drugs on this protein). Zonal elution competition studies were used to examine the interactions of myristic acid, palmitic acid and stearic acid with these probes on HSA. It was found that all these fatty acids had direct competition with R-warfarin at Sudlow site I of normal HSA and glycated HSA, with the glycated HSA typically having stronger binding for the fatty acids at this site. At Sudlow site II, direct competition was observed for all the fatty acids with L-tryptophan when using normal HSA, while glycated HSA gave no competition or positive allosteric interactions between these fatty acids and L-tryptophan. These data indicated that glycation can alter the interactions of drugs and fatty acids at specific binding sites on HSA. The results of this study should lead to a better understanding of how these interactions may change during diabetes and demonstrate how HPAC can be used to examine drug/solute-protein interactions in complex systems.