目的研究人白蛋白与3-氨甲基吡啶(潜在的亲和配体)之间的相互作用规律。方法在不同酸碱度和盐浓度的缓冲液条件下将固定化3-氨甲基吡啶与人血浆中的白蛋白接触，测定3-氨甲基吡啶对人白蛋白的结合量。用亲和毛细管电泳方法测定了液相状态下白蛋白与3-氨甲基吡啶相互作用的亲和常数及结合位点的数目，并与固相状态下的作用机制进行了比较。最后用计算机辅助分子模拟的方法，在白蛋白三级结构上搜寻可能的结合位点， 结合实验数据阐明相互作用的机制。结果在pH8.0时结合量最高，并随pH升高和降低而减少；结合的量随盐浓度的增大而减少,潜在结合口袋区域存在ASP256,ASP259两个带电荷的氨基酸残基。结论静电相互作用是白蛋白与3-氨甲基吡啶的结合的主要动力，吡啶环的疏水性质和空间体积的排斥性质可能决定了白蛋白与3-氨甲基吡啶相互作用的专一性。
Abstract
OBJECTIVE To study the interaction between human albumin and the affinity ligand 3-amino methyl pyridine. METHODS Immobilized 3-aminomethyl pyridine was contacted with albumin in human plasma under buffered solutions at different pHs and concentrations of salt.The amount of albumin bound was analyzed.In addition,the affirity constants of human albumin and the free ligand and immobilized ligand were measured.Computer aided molecular modeling was used to search the potential binding site on the surface of albumin,and the interaction was scrutinized with the properties of functional chemical groups at the binding site of amino acid. RESULTS Albumin binding capacity reached peak at pH8.0,and decreased with pH increasing or decreasing.The binding capacity decreased when increasing salt concentration.Two charged groups of ASP256 and ASP259 at the potential binding site were found. CONCLUSION Electrostatic interactions appeared to be the primary interactions between human albumin and 3-amino methyl pyridine.Hydrophobic nature and steric hindrance of pyridine probably contribute to the recognition specificity.
关键词
结合位点 /
相互作用 /
白蛋白 /
三氨甲基吡啶 /
亲和常数
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Key words
binding sites /
interactions /
albumin /
3-amino methyl pyridine /
association constant
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参考文献
[1]Villamor JP,Zaton AML.Data plotting of wafarin binding to human serum albmin[J]. J Biochem Biophys Methods, 2001,48:33.
[2]Bertucci C,Andrisano V,Gotti R, et al. Use of immobilized human serum albumin HPLC column as a probe of drug-protein interactions:the reversible binding of valproate[J]. J Chromatogr B, 2002,768:147.
[3]Chaiken IM,Wilchek M,Parikh I. Affinity chromatography and biological recognition, 1983:8～9.
[4]汪家政，范明.蛋白质技术手册[M].北京:科学出版社，2000：77～110.
[5]Kiessig S,Thuneche F.Investigation of cyclophilin interactions with oligopeptides containing proline by affirity capillary electrophoresis[J]. J Chromatogr A, 2002,982:275.
[6]Teng SF,Sproule K,Husain A, et al. Affinity chromatography on immobilized “biomimetic” ligands Synthesis,immobilization and choromatographic assessment of an immunoglobulin G-binding ligand[J]. J Chromatogr B, 2000,740:1.
[7]周绪斌，宣华，李荣秀.组特异性亲和配基在分离纯化中的作用[J].中国生化药物杂志，2000，21(6)：308.
[8]Rongxiu LI.An artificial protein A.Ph.D. Thesis in University of Cambridge, 1995:99～109.
[9]Gomez FA,Mirkovich JN,Dominguez VM, et al. Multi-plug binding assays using affirity capillary electrophoresis[J]. J Chromatogra A, 1996,727:291.
[10]Shimura K,Kasai KI.Affinity capillary electrophoresis:a sensitive tool for the study of molecular interaction and its use in microscale analyses[J]. Anal Biochem, 1997,251:1.
[11]Curry S,Brick P,Franks NP.Fatty acid binding to human serum albumin: new insights from crystallographic studies[J]. Biochim Biophys Acta, 1999,1441:131.
[12]Bhattacharya AA,Curry S,Franks NP.Binding of the general anesthetics proposal and halothane to human serum albumin.high resolution crystal structures[J]. J Biol Chem V, 2000,275:38731.
[13]Chattopadhyay A,Tian T,Kortum L, et al. Development of tryptophan-modified human serum albumin columns for site-specific studies of drug-protein interactions by high-performance affirity chromatography[J]. J Chromatogr B, 1998,715:183.
[14]Tuan Giam Chuang V,Kuniyasu A,Nakayama H, et al. Helix 6 of subdomain IIA of human serum albumin is the region primarily photolabeled by ketoprofen,on arylpropionic acid NSAID containing a benzophenone moiety[J]. Biochim Biophys Acta, 1999,1434:18.
[15]Ana Mary′a Lourdes Zato′n,Villamor JP.Study of heterocycle rings binding to human serum albumin[J]. Chem Biol Interact, 2000,124:1.
[16]Tuan Giam Chuang V,Flunitrazepam MO.A 7-nitro-1,4-benzodiazepine that is unable to bind to the indole-benzodiazepine site of human serum albumin[J]. Biochim Biophys Acta, 2001,1546:337.
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脚注
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基金
国家自然科学基金资助项目(39980032)；国家高技术研究发展计划经费资助项目(Z21-04-05和103-13-03-07)
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